A Study on Tumor Suppressor Genes Mutations Associated with Different PPPathologicalPathological CCColorectalColorectal LLLesions.Lesions.
Thesis Submitted for partial fulfillment of the requirements for the Ph.D. degree of Science in Biochemistry
By Salwa Naeim Abd ElEl----KaderKader Mater M.Sc. in Biochemistry, 2002
Biological Applications Department Nuclear Research Center Atomic Energy Authority
Under the supervision of
Prof. Dr. Amani F.H Nour El ---Deen Prof. Dr. Abdel Hady Ali Abdel Wahab Professor of Biochemistry Professor of Biochemistry Biochemistry Department and Molecular Biology Faculty of Science Cancer Biology Department Ain Shams University National Cancer Institute Cairo University
Prof. DrDr.MohsenMohsen Ismail Mohamed Dr. Azza Salah Helmy
Professor of Clinical Pathology Biological Assistant Professor of Biochemistry Applications Department Biochemistry Department Nuclear Research Center Faculty of Science Atomic Energy Authority Ain Shams University
2012011111
ﺑﺴﻢ ﺍﷲ ﺍﻟﺮﺣﻤﻦ ﺍﻟﺮﺣﻴﻢ
ﺇِﻧﻤﺎ ﺃﹶﻣﺮﻩ ﺇِﺫﹶﺍ ﺃﹶﺭﺍﺩ ﺷﻴﺌﹰﺎ ﺃﹶﻥﹾ ﻳﻘﹸﻮﻝﹶ ﻟﹶﻪ ﻛﹸـﻦ ﻓﹶﻴﻜﹸـﻮﻥﹸ ””” ٨٢“٨٢٨٢٨٢“““ ﻓﹶﺴﺒﺤﺎﻥﹶ ﺍﻟﱠﺬِﻱ ﺑِﻴﺪِﻩِ ﻣﻠﹶﻜﹸﻮﺕ ﻛﹸـﻞﱢ ﺷـﻲﺀٍ ﻭﺇِﻟﹶﻴـﻪِ ﺗﺮﺟﻌﻮﻥﹶ ””” ٨٣“٨٣٨٣٨٣“““ ﺻﺪﻕ ﺍﷲ ﺍﻟﻌﻈﻴﻢ رة ( ٨٢ )٨٣
I declare that this thesis has been composed by myself and the work there in has not been submitted for a degree at this or any other university.
Salwa Naeim Abd El Kader Matter
To my dear husband and family Their love, encouragement and help made my studies possible and to them I owe everything.
Thank you
Salwa Naeim Abd El Kader Matter
Acknowledgement
First of all, thanks to Allah the most merciful for guiding me and giving me the strength to complete this work.
It is pleasure to express my deepest thanks and profound respect to Prof. Dr. Amani F.H Nour El-Deen , Professor of Biochemistry Faculty of Science Ain Shams University, for her continuous encouragement, valuable supervision and guidance through this work.
Also, I wish to express my deepest gratitude to Dr. Azza Salah Helmy , Assistant professor of Biochemistry Faculty of Science Ain Shams University, for her valuable suggestions, sincere guidance and support. I shall be unlimited thankful for her.
I am also deeply grateful and would like to express my sincere thanks and gratitude to Prof. Dr. Abdel Hady Ali Abdel Wahab , Professor of Biochemistry and Molecular Biology , Cancer Biology Department National Cancer Institute Cairo University, for his great help, valuable assistance and his continuous guidance which helping me to finish this work.
I would like to express my sincere thanks and gratitude to Prof. Dr. Mohsen Ismail Mohamed, Professor of Clinical Pathology, Nuclear Research Center Atomic Energy Authority, for his great help, valuable assistance and for sparing his valuable time and effort to review this work. I shall be unlimitedly thankful for him.
I would like to express my sincere thanks to Prof. Dr. Laila Ghazy Abd El-Khalek, Professor of Physiology, Nuclear Research Center Atomic Energy Authority, for her incredible and valuable assistance.
Sincere gratitude is directed to all staff members and my collages of Clinical Chemistry Unit Nuclear Research Center Atomic Energy Authority, for their incredible and valuable assistance.
Sincere gratitude is directed to my colleagues in Cancer Biology Department National Cancer Institute Cairo University for their faithful support and helpful to me in the laboratory techniques.
Last but not least, I would like to thank my family, for their kind support during accomplishment of this work to bring it into its current state.
Biography
Name : Salwa Naeim Abd El Kader Matter
Date of graduation : B.Sc (Biochemistry), 1995 M.Sc (Biochemistry), 2002, Faculty of Science, Ain Shams University .
Occupation : Assistant Lecturer, Nuclear Research Center, Atomic Energy Authority
Supervisors:
Prof. Dr. Amani F.H Nour El-Deen
Prof. Dr. Abdel Hady Ali Abdel Wahab
Prof. Dr. Mohsen Ismail Mohamed
Dr. Azza Salah Helmy
List of Abbreviations
ACF aberrant crypt foci AFP Alpha Fetoprotein AP adenomatous polyps APC Adenomatous polyposis coli gene APS Ammonium persulfate bp Base pair BTEB1 basic transcription factor element binding protein BTEB2 basic transcription factor element binding protein 2 CD Crohn's disease CEA Carcinoembryonic Antigen CIN Chromosomal instability COPEB core promoter element binding protein CPBP core promoter binding protein CPBP Core Promoter Binding Protein CPE core promoter element CRC Colorectal cancer DCBE Double contrast barium enema DCC Deleted in colorectal cancer gene ddH 2O Deionized distilled water DNA Deoxi ribonucleic acid DRE Digital rectal exam EDTA Ethylene Diamin tetraacetate ER Estrogene receptor FAP familial adenomatous polyps FKLF fetal erythroid Kruppel like factor FOBT Fecal occult blood test Ha MSV Harvey murine sarcoma viruses IBD Inflammatory Bowel Disease IRB institutional review board Kd Killo Dalton Ki MSV Kirsten murine sarcoma viruses KLF6 Krüppel like factor 6 KLF family Krüppel like transcriptional factors family Lis SSCP low ionic strength Single strand conformation loading buffer polymorphism loading buffer LOH Loss of heterozygosity MMR DNA mismatch repair MSI micro satellite instability OD Optical density PCR LIS polymerase chain reaction low ionic strength SSCP silver Single strand conformation polymorphism silver staining staining method PSA Prostate Specific Antigen PSG Pregnancy Specific Glycoprotein genes PSG pregnancy specific glycoprotein SNP Single nucleotide polymorphism SSCP Single strand conformation polymorphism analysis TAE Tris Acetate EDTA buffer TBE Tris Borate EDTA buffer TE buffer Tris EDTA buffer TEMED N,N,N /,N / tetramethylene diamine TGF β transforming growth factors β U.C ulcerative colitis UC CRC Ulcerative colitis colorectal cancer pathway pathway UKLF ubiquitous Kruppel like factor UTR Un translated regions uPA urokinase type plasminogen activator 10p15 Short arm of chromosome 10 (locus 15)
List of Tables
TABLE TITLE PAGE NO.
1 The clinical data for the patients of adenocarcinoma group (I)…………………… 48
2 The clinical data for the patients of polyps group (II)……………………………………. 49
3 The clinical data for the patients of IBD group (III)…………………………………… 50
4 Primer sequences and the size range for klf6 exones used………………………………….. 57 5 PCR conditions for KLF6 gene (exons 1 4) amplifications……………………………….. 58
6 The primer sequences and the size of the amplified fragments for the KLFM1, KLFM2 and KLFM4 markers………………………… 67
7 PCR conditions for KLF6 gene (LOH markers) amplifications…………………… 68
8 The pattern of mutations, codon no. and amino 82 acid changes in the examined colon cancer cases (GI).
9 The pattern of mutations, codon no. and amino acid changes in the examined colon polyp cases (GII)……………………………………………. 85
10 The pattern of mutations, codon no. and amino acid changes in the examined colon IBD cases (GIII)…………………………………………… 88
11 The clinicopathological data of some cases in GI……………………………………………. 104
12 Statistical relations in GI……………………. 104
13 The clinicopathological data of some cases in GII…………………………………………… 105 14 The clinicopathological data of some cases in GIII…………………………………………… 107
List of Figures
FIGURE TITLE PAGE NO.
1 Colon anatomy……………………………..... 12
2 Diagram shows the progress of Colon cancer start from colon polyps………………………. 16
3 Colorectal anatomy diagram show different lesions and cancer anatomy………………….. 17
4 Schematic diagram for different colorectal cancer stages………………………………. 19
5 Proposed adenoma to carcinoma sequence in colorectal cancer… …………………….. 29
6 Schematic representation of the putative steps in colorectal cancer progression……... 36
7 KLF6 protein structure…………………….. 41
8 Functional domains of KLF6 protein……… 42
9 Location of KLF6 gene on chromosome 10. 43
10 Genomic structure of KLF6 gene…………. 44
11 A schematic representation of the SSCP technique………………………………….. 56
12 Genomic DNA isolated from tissue biopsies for cases no. 1 8 of the CRC group. ………. 72 13 The percentage distribution for KLF6 mutation in the three different studied groups……………………………………… 73
14 The percentage distribution for mutations in the two age categories in the three examined groups………………………………………. 74 15 The percentage distribution for mutations in the males and females in the three examined groups……………………………………… 75
16 The percentage distribution for mutations of KLF6 gene in different lesion sites in the three examined groups…………………….. 76
17 The percentage distribution for mutations of KLF6 gene in different pathological grades of colon cancer cases in cancer group. ……. 77
18 The percentage distribution for mutations of KLF6 gene in the two categories of ulcerative colitis cases……………………... 78
19 The percentage distribution for mutations of 79 KLF6 gene in the two size categories of polyps group.
20 A The percentage distribution for mutations of 80 different exons of KLF6 gene in cancer group (GI).
20 B The percent of mutations for KLF6 gene exons according to sequencing results in cancer group. ……………………………... 81
21 A Detection of KLF6 gene mutations in CRC B sample no. 1. (A) SSCP analysis for exon 2 for CRC samples (T1 T5)…………………………… 83 (B) Direct sequence analysis representative sample (case no. 1) of CRC for both normal and tumor tissues………… 83
22 A The percentage distribution for mutations of different exons of KLF6 gene in Polyps group (GII)………………………………… 84
22 B The percentage of mutations for KLF6 gene exons according to sequencing results in polyps group……………………………….. 85
23 SSCP silver staining analysis for exon 2 of KLF6 gene for polyps tissues. ……………. 86
24 A The percentage distribution for mutations of different exons of KLF6 gene in IBD group……………………………………….. 87
24 B The percentage of mutations for KLF6 gene exons according to sequencing results in IBD group………………………………….. 88
25 SSCP silver staining analysis for exon 1 of KLF6 gene for IBD tissues………………… 89
26 PCR product for the 3 markers used in the (A,B&C) present study (KLFM1, KLFM2 and KLFM4) without using radioactive 32 P in the reaction………………………………… 91
27 Percentage of LOH in cancerous group for the 3 different microsatellite markers used... 92
28 LOH study in cancerous group for KLFM1 marker……………………………………….. 92
29 Individual distribution of LOH analysis in cancer group for the 3 microsatellite markers examined…………………………………….. 93
30 The percentage distributions of LOH markers with different clinicopathological examined factors………………………………………… 94
31 Percentage of LOH in IBD group for the 3 different microsatellite markers used………… 95
32 LOH study in IBD group for KLFM4 and KLFM2 markers. ……………………………. 96
Individual distribution of LOH analysis in 33 IBD group for the 3 microsatellite markers examined…………………………………….. 97
34 The percentage distributions of LOH markers with different clinicopathological examined factors……………………………………….. 98
35 Percentage of LOH in polyps group for the 3 different microsatellite markers used………… 99
36 LOH study in polyps group for KLFM4 and KLFM2 markers. ……………………………. 100
37 Individual distribution of LOH analysis in 101 polyps group for the 3 microsatellite markers examined.
38 The percentage distributions of LOH markers with different clinicopathological examined factors………………………………………… 102
39 The distribution of KLF6 status results in GI 103
40 The distribution of KLF6 status results in GII.. 105
41 The distribution of KLF6 status results in GIII…………………………………………… 106
Contents
Abstract …………………………………………………………………….. i Introduction …………………………………………………………….…. 1
Aim of the work ………………………………………………………… 6 Review of Literature ………………………………………………….. 7 An overview on cancer I- ………………………………….. 7 • Cancer definition …………………………………………………. 7 • Benign and malignant tumors ………………………….……. 7 • Causes of Cancer……………………………………...…………… 9 II COLORECTAL LESSIONS AND CANCER …………… 11 (1) Anatomy of the colon ……………………………………………... 11 (2) Colorectal lesions…………………………………………….……… 13 (3) Colorectal cancer………………………………...………………… 17 (a) Colorectal cancer progress ………………………………...... 18 (b) The symptoms of colorectal cancer…………………………….. 20 (c) Risk factors of colorectal cancer…………………………..…….. 20 (d) Methods of colorectal cancer screening…………………..……... 21 (4) Epidemiology of colorectal lesions and cancer………………….… 25 (5) Colorectal carcinoma in Egyptian patients………………………. 27 (6) Molecular pathways of colorectal cancer……………………..….. 28 III- Gene involved in the pathways of colorectal cancer……..………. 30
IV --- KLF 6 tumor suppressor gene …………………….………… 37 (1) Introduction……………………………………………………. 37
(2) KLF6 protein…………………………………………………… 40
(3) KLF6 gene nomenclature and identification…………………. 43
(4) KLF 6 tumor suppressor gene and Cancer…………………… 45
Subjects and Methods ……………………………..………………… 47 Results ………………………………………………….…………….. 72 Discussion ……………………………………………………..………. 108 Summary ………………………………………………………………. 120 References …………………………………………………………..…. 123 Arabic summary ………………………………………………..……..
Abstract
Colorectal cancer (CRC) is the second leading cause of cancer related death in the Western world. In Egypt; there is an increasing incidence of the disease, especially among patients ≤40 years age. While CRC have been reported in low incidence rate in developing countries , it is the third most common tumor in male and the fifth common tumor in females in Egypt. Early diagnosis and surgical interference guarantee long survival of most CRC patients. Early diagnosis is impeded by the disease onset at young age and imprecise symptoms at the initial stages of the disease. As in most solid tumors, the malignant transformation of colonic epithelial cells is to arise through a multistep process during which they acquire genetic changes involving the activation of proto oncogenes and the loss of tumor suppressor genes. Recently, a candidate tumor suppressor gene, KLF6, which is mapped to chromosome 10p, was found to be frequently mutated in a number of cancers. There are some evidences suggesting that the disruption of the functional activity of KLF6 gene products may be one of the early events in tumorgenesis of the colon. The main objective of the present study was to detect mutational changes of KLF6 tumor suppressor gene and to study the loss of heterozygosity (LOH) markers at chromosome 10p15 (KLF6 locus) in colorectal lesions and colorectal cancer in Egyptian patients. The patients included in this study were 83 presented with different indications for colonoscopic examination. Selecting patients with colorectal pre cancerous lesions or colorectal cancer was done according to the results of tissue biopsy from lesion and adjacent normal. The patients were classified into three main groups; (GI) Cancerous group, (GII) polyps group including patients with adenomatous polyps (AP), familial adenomatous polyps (FAP) and hyperplastic polyps (HP) and (GIII) Inflammatory Bowel Diseases (IBD) including patients with ulcerative colitis (UC) and Crohn's disease (CD). Purified DNAs which were extracted from the tissue samples, PCR amplified and subjected to the following examinations: 1- Detection of KLF6 mutations by using SSCP silver staining technique and DNA sequencing by using BigDye Terminator v3.1Cycle Sequencing kit using Biosystem automated sequencer (The ABI PRISM 3100 Genetic Analyzer). 2- Determination of Loss of heterozygosity (LOH) on chromosome 10p15 regions (KLF6 locus) by using three microsatellite markers which includes KLFM1, KLFM2, and KLFM4. Data from the present study could be
i summarized as follows: In GI, 55.3% of cases had abnormalities in KLF6 gene (mutations and LOH). LOH was detected in 29% of investigated samples while KLF6 mutations were detected in 44% of cases. In GII, 57% of cases had abnormalities in KLF6 gene (mutations and LOH). LOH was detected in 55% of investigated samples while mutations of KLF6 gene were detected in 26% of investigated samples. In GIII, 50% of samples had abnormalities in KLF6 gene (mutations and LOH). LOH was detected in 36.4% of investigated samples while mutations of KLF6 gene were detected in 27.3% of investigated samples. Most of the mutations reported were of the missence and /or Transversion type and were almost in exon 2. In conclusion, our data highlight for the first time a role of KLF6 gene in the progression of Egyptian colorectal carcinogenesis where the results suggest that KLF6 gene alteration is involved in the progression of Egyptian colorectal carcinogenesis from both sporadic adenomatous polyps and ulcerative colitis pathways. Detecting mutational sites differing from that detected in western populations may be a characteristic of Egyptian CRC due to environmental and genetic factors. The detections of such genetic abnormalities may also be used as a marker for the early uncovering of colon cancer cases. It is recommended that those who have pre neoplastic colon lesions in which the KLF6 gene has mutated or lost its heterozygosity should experience more frequent colonoscopic examinations for the detection of doubtful malignant changes. The present study also paves the way to further research needed to elucidate the possible role of KLF6 protein in the transactivation of other genes involved in cell cycle regulations and apoptosis. It also supports the possibility of using KLF6 gene as a target for gene therapy in colorectal cancer.
ii Introduction
INTRODUCTION AND AIM OF THE WORK
Introduction:
Normal development is a balance process, which includes proliferation and cell death. Indeed both proliferation and apoptotic cell death are very complex process that involves the participation of many genes. In both events, the tumor suppressor genes are the most important and studied genes (Jung & Messingm, 2000). The carcinogenic procedure is a multistep process involving several genetic alterations that eventually ends in malignant transformation. The study of carcinogenic procedure is very important, because it does not only shed light on some critical steps in the progress of carcinogenesis but may also provide a rational approach for early diagnosis of cancer (Mendoza-Rodriguez & Cerbon, 1995).
Although some diagnostic markers are available that are assayable from blood or tissue samples, e. g. Carcinoembryonic Antigen (CEA), Alpha Fetoprotein (AFP) or Prostate Specific Antigen (PSA), the assays using these markers have not, to date, been markedly predictive of the presence of cancer in these individuals, as verified by other clinical diagnoses. The sensitivity and specificity of these assays has been disappointingly low. Time consuming and labor intensive clinical assessments (e. g. palpations, x rays, mammograms, biopsies) have remained the accepted methods for diagnosing cancer. Thus, a need exists for a biomarker that is predictive of the presence of cancer or of an increased risk of developing a cancer in the individual. In particular, a need exists for a marker and an assay to measure the presence and amount of this marker for individuals with an early stage of cancer. If such diagnostic test is available, early treatment with beneficial outcomes would be more likely than at present (Srivastava and Gopal- Srivastava , 2002).
One of the most important discoveries in cancer biology was the cancer arise as a result of cumulative genetic changes in cells. The progression of a tumor from normal cells to pre cancerous ones, to cancer and then to local invasion and finally metastasis is the result of the clonal expansion of cells that have acquired a selective growth advantage, which allows them to outnumber neighboring cells. This
1
Introduction advantage is the result of changes in genes that control cellular proliferation and cell death (Nowell, 1976).
Researchers have found the first connection between the loss of a tumor suppressor genes and activation of cancer promoting oncogenes, a scenario thought to be prevalent in the initiation of many cancers but which has never been proved . Several types of cancers have been shown to be associated with the loss of function of a tumor suppressor. Tumor suppressor genes were first identified by making cell hybrids between tumor and normal cells (Vogelestien et al., 2000).
Clinical screening is the testing of apparently asymptomatic individuals to classify them into those who are likely to have disease and those who are not; that are the detection of disease in its pre clinical phase (Hruban et al., 1993). The possibility of using molecular markers to detect cancer in the pre clinical phase has been demonstrated by several case reports (Wakabayashi et al., 1996).
Colorectal cancer (CRC) is the second leading cause of cancer related death in the Western world (Weitz et al., 2005). In countries located in Eastern Asia like Korea, it accounts for an estimated 11.2% of all malignancies; 11.6% in the male population and 10.7% in the female population (Shin et al., 2004). Thus, colorectal cancer remains a significant contributor to the world’s health burden. Considerable progress has been made with respect to this disease at a molecular level in the last two decades via identification and characterization of the genetic changes involved in the malignant colorectal transformation process. Thus, the concept of multistage carcinogenesis is now widely accepted as being a consequence of multiple genetic alterations that are accumulated in cancer cells . However, the molecular mechanisms underlying the dysregulated cell growth in colorectal cancer remain the subject of intense investigation (Fearon and Vogelstein., 1990).
In Egypt, there is an increasing incidence of colorectal cancer, especially among patients' ≤40 years of age, whereas CRC have been reported in low incidence rate in developing countries (EL-Hennawy et al., 2003). It’s the third most common tumor in male after urinary bladder and lymphohemopoietic malignancies and in females it ranks fifth after breast, lymphohemopoietic, cervical and bladder cancers (Abou-Zeid et al., 2002). Westernization of Egypt is occurring as the
2
Introduction country develops and is affecting young people first because they are likely to change lifestyle than older people. The remarkable differences in molecular pathology as compared to western patients could result from inherent differences in sensitivity and molecular responses to western lifestyle in Egyptians (Rockhill and Giovannucci, 1999). Another study on CRC in Egyptian patients revealed that colorectal cancer in Egypt has no age predilection and more than one third of tumors affect a young population. The high prevalence in young people can neither be explained on a hereditary basis nor can it be attributed to bilharziasis. The disease usually presents at an advanced stage and predisposing adenomas are rare. Also, similarity of the data from different centers suggests that this is the picture of colorectal cancer typical of Egyp t (Abou-Zeid et al., 2002).
The molecular pathology of colorectal carcinoma in Egypt differs from western patients, and between younger and older Egyptians (Soliman et al., 2001). Although Arabic countries share some culture background and environmental exposures (Temtamy et al., 1994 and Al-Saleh et al., 1998), rates of young onset disease are higher in Egypt than Jordan (Al-Jaberi et al., 1997), Lebanon (Adib et al., 1998), Algeria (Parkin et al., 1992) and Saudi Arabia (Isbister.,1992).
The rectal predominance in Egyptian cases occurred in both younger and older patients and was unrelated to urban/rural residence, which associated with very different environmental exposure (Soliman et al., 2001). Also rectal cancer patients <40 years of age have more advanced disease at presentation and a higher incidence of treatment failure caused by both a delay in the diagnosis and a more aggressive pattern of the disease (EL-Hennawy et al., 2003).
Studies on colorectal cancers in Egypt showed a high proportion of cases occurring before age 40 years. Unusual pathologic features in this population include few K ras mutations (11 %), high frequency of rectal tumors (51 %), and poorly differentiated histology (58%) and high levels of micro satellite instability (MSI H, 37%) (Chan, On-on, Annie., 2002). Studying of unusual cancer distribution and analysis of particular genetic mutations may provide clues to cancer etiology (Soliman et al., 2006).
3
Introduction
Krüppel like factor 6 (KLF6) is a ubiquitously expressed zinc finger transcription factor that is part of a growing KLF family. The KLF gene family is broadly involved in cell differentiation and development, growth related signal transduction, cell proliferation, apoptosis and angiogenesis (Bieker.2001, Black et al., 2001).There is a growing body of evidence that KLF6 gene can act as atumor suppressor gene. The genetic alterations of the KLF6 gene have recently been identified in several human cancers, including hepatocellular carcinoma, prostate, gastric and colon cancers (Chen et al., 2003, Kremer-Tal et al., 2004, Reeves et al., 2004; Boyault et al., 2005& Cho et al., 2005). As observed on functional analysis, wild type KLF6 up regulates the cell cycle inhibitor p21 in a TP53 independent manner and it suppresses growth, whereas tumor derived KLF6 mutants fail to upregulate p21 or suppress proliferation in prostatic and non small cell lung cancer cells (Narla et al., 2001& Ito et al., 2004). In addition, introduction of KLF6 disrupts the cyclin D1 cyclin dependent kinase 4 complexes and forces the redistribution of p21, which promotes G1 cell cycle arrest (Benzeno et al., 2004). KLF6 also plays a role as an inhibitor of cell proliferation by counteracting the function of c Junc protooncoprotein (Salvin et al., 2004). These findings have led to the hypothesis that genetic alteration of the primary structure of the KLF6 gene might be one of the possible mechanisms of KLF6 inactivation in colorectal cancers.
Allelotype studies have shown that specific chromosome regions are frequently deleted in many tumor types and the frequent loss of heterozygosity (LOH) at certain chromosomal regions is a hallmark of the existence of a tumor suppressor gene. Therefore, identifying consistent areas of chromosomal deletion in a particular tumor’s DNA may indicate regions that harbor tumor suppressor gene at this locus, which contributes to carcinogenesis in that tissue. In a previous allelotype analysis on all the chromosomes of colorectal cancer, frequent LOH has been reported on the short arm of chromosome 10 (Shima et al., 2005). Because the KLF6 gene is located on chromosome 10p15 (Onyango et al., 1998), it may be one of the possible tumor suppressor genes in human cancers.
Cancer progresses through a series of histopathological stages. Progression is thought to be driven by the accumulation of genetic alterations and consequently gene expression pattern changes. The identification of genes and pathways involved will not only enhance our understanding of the biology of this process, it will also
4
Introduction provide new screening targets for those who are more likely to develop cancer (Garnis et al., 2004).
The increased risk of colorectal cancer has been supposed to be a result of shared genetic susceptibility. Rhodes (1996) has suggested the existence of a genetically mediated glycosylation defect, which would be of importance in the development of cancer and IBD. In support of this assumption, Askling et al (2001) had demonstrated that relatives of patients with both IBD and colorectal cancer had an 80% increased risk of colorectal cancer.
There are also some evidences that the KLF6 gene mutations may play a role in the early genetic changes that accompany carcinogenesis of the colon. Reeves et al (2004) have reported that the pattern of KLF6 loss in inflamed colonic epithelium was similar to that seen in the adjacent tumor, which suggested that KLF6 loss may be an early event in the pathogenesis of colorectal cancer. In the mean time , Ito et al (2004) had reported that in non small cell lung cancer, there was no association between KLF6 expression and clinicopathological parameters.
5
Introduction
Aim of the work
The main objective of the present study is to detect mutational changes of KLF6 tumor suppressor gene and some loss of heterozygosity (LOH) markers at chromosome 10p15 (KLF6 locus) in both established colon cancer and in colon lesions with increased risk of developing colon cancer cases. The later included adenomatous polyps (AP), familial adenomatous polyps (FAP), hyperplastic polyps (HP) lesions and inflammatory Bowel diseases associated lesions. Defining the genetic changes in KLF6 locus in the pre neoplastic conditions may highlight cases who should experience more frequent colonoscopic examinations for the early detection of suspicious lesions. In addition, studying the genetic alteration that accompanies the carcinogenic transformation is very important. It does not only shed light on some critical steps in the progression of cancer but may also provide a rational approach for early diagnosis of cancer.
6
Review of literature
REVIEW OF LITERATURE
I- An overview on cancer
● Cancer definition :
Cancer is not one disease, but many diseases that occur in different areas of the body. Each type of cancer is characterized by the uncontrolled growth of cells. Under normal conditions, cell reproduction is carefully controlled by the body. However, these controls can malfunction, resulting in abnormal cell growth and the development of a lump, mass or tumor. Some cancers involving the blood and blood forming organs do not form tumors but circulate through other tissues where they grow. (American cancer society,
Inc, 2002)
A tumor may be benign (non cancerous) or malignant (cancerous). Cells from cancerous tumors can spread throughout the body. This process, called metastasis, occurs when cancer cells break away from the original tumor and travel in the circulatory or lymphatic systems until they are lodged in a small capillary network in another area of the body. Common locations of metastasis are the bones, lungs, liver and central nervous system. (American cancer society, Inc, 2002)
The type of cancer refers to the organ or area of the body where the cancer first occurred. Cancer that has metastasized to other areas of the body is named for the part of the body where it originated. For example, if breast cancer has spread to the bones, it is called "metastatic breast cancer" not bone. (American cancer society, Inc, 2005)
● benign and malignant Tumors:
Benign tumors are not cancer. Usually, doctors can remove them. Cells from benign tumors do not spread to other parts of the body. In most cases, benign tumors do not come back after they are removed. Most important, benign tumors are rarely a threat to life (Fadl Elmula et al., 2002).
7
Review of literature
Malignant tumors are cancer. They are generally more serious. Cancer cells can invade and damage nearby tissues and organs. Also, cancer cells can break away from a malignant tumor and enter the bloodstream or the lymphatic system. That is how cancer cells spread from the original (primary) tumor to form new tumors in other organs. The spread of cancer is called metastasis (Fadl Elmula et al., 2002).
Cancers or malignant tumors are classified according to the type of tissue from which they originate. The broadest division of cancers separates the carcinomas, tumors which arise from epithelial tissues, and the sarcomas, which arise from all other tissues. Epithelium is tissue that covers the internal or external surfaces of the body. Thus, skin, the lining of the mouth, stomach, intestines and bladder and so on are all epithelial tissue (Rubin, 2001).
Within the category of carcinomas, there are many subdivisions, corresponding to the types of different epithelium from which they may be derived. Therefore, the skin, which consists of a type of epithelium called squamous epithelium, can give rise to squamous cell carcinomas. There are other epithelial cells also present in the skin, basal cells, which give rise to basal cell carcinomas and melanocytes, which give rise to melanomas (Rubin, 2001).
Adenocarcinoma is a cancer originating in glandular cells. Adenocarcinoma occurs in the lungs, from small glands in the bronchi; in the stomach from one of the several types of glands lining it; and in the colon, breast, ovaries, testes, prostate and in other locations. Adenocarcinoma arising from different organs can often be identified by the pathologist microscopically, even when they are removed from a different location where they may have metastasized, such as the liver. Thus, it is common to refer to an adenocarcinoma of the stomach which has metastasized to the liver, or one from the colon metastasized to the lungs. Adenocarcinoma are the most common cell type of cancer, since they include almost all breast cancers, all colon cancers, all prostate cancers and a fair percentage of lung cancers (Rubin., 2001).
8
Review of literature
● Causes of Cancer:
Ninety percent of cancers develop because of complex interactions between our bodies, our lifestyles, our genetic makeup and our environment. Scientists have discovered different factors that cause cancer. Research shows that tobacco is estimated to cause 30 percent of all cancer deaths, poor diet 35 percent, reproductive and sexual behavior seven percent, work related causes four percent and the environment itself causes three percent (American cancer society, Inc, 2005). So, cancers are caused by:
**Anything that damages DNA and called mutagenic such as:
1 Radiation that can penetrate to the nucleus and interact with DNA. Sources of ionizing radiation, such as radon gas, can cause cancer. Prolonged exposure to ultraviolet radiation from the sun can lead to melanoma and other skin malignancies (Armstrong et al., 1997). Non ionizing radiation from mobile phones and other sources has also been proposed as a cause of cancer (Feychting et al., 2005).
2 Chemicals that can penetrate to the nucleus and damage DNA. Chemicals that cause cancer are called carcinogens.
●Tobacco smoke: smoking developed cancer of the lung, mouth, throat, esophagus, bladder and pancreas. Smoking is thought to cause about a quarter of all cancers. About 1 in 10 smokers die from lung cancer. ●Workplace chemicals such as asbestos, benzene, formaldehyde, etc. and working with these without protection will increase risk of developing certain cancers. For example, a cancer called mesothelioma is linked to past exposure to asbestos (Pazdur et al., 2007 and Bast et al., 2000).
**Anything that stimulates the rate of mitosis such as:
1 Certain hormones : Some hormones can act in a similar manner to non mutagenic carcinogens in that they may stimulate excessive cell growth. A well established example is the role of hyperestrogenic states in promoting endometrial cancer. Also hormones that stimulate mitosis in tissues like the breast and the prostate gland (WHO, 2006). 9
Review of literature
2 Chronic tissue injury : In which increases mitosis in the stem cells needed to repair the damage (American cancer society, Inc, 2002).
3 Agents that cause inflammation: At which generates DNA damaging oxidizing agents in the cell (American cancer society, Inc , 2002)
4 Viruses: Many viruses have been studied that reliably cause cancer when laboratory animals are infected with them and the term an oncovirus refers to the virus associated with cancer such as the following examples:
The hepatitis B and hepatitis C viruses, which infect the liver and are closely associated with liver cancer probably because of the chronic inflammation they produce (Hu and Ludgate, 2007).
Some herpes viruses such as the Epstein Barr virus implicated in Burkitt's lymphoma and KSHV that is associated with Kaposi's sarcoma; a malignancy frequently seen in the late stages of AIDS) (Chang et al., 1994).
Two human T cell leukemia viruses, HTLV 1 and HTLV 2. The viral infection only contributes to the development of cancer but many people are infected by these viruses and do not develop cancer (Bellon and Nicot, 2007).
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II- COLORECTAL LESSIONS AND CANCER
(1) Anatomy of the colon
The colon is the last part of intestines and is also known as the large intestine. The locations of the parts of the colon are either in the abdominal cavity or behind it in the retroperitoneum. The colon in those areas is fixed in location. Swallowed food goes through the esophagus, also known as "the feeding tube". It then passes through the stomach, where it is partially digested. Digested food goes from the stomach to the small intestines, where nutrients are further digested and absorbed. Fibers and digested food then reach the colon. In the colon, the rest of the nutrients get absorbed and stool are formed. Stool are then stored in the last part of the colon, the sigmoid and rectum, before being excreted (Bowen et al., 2007 and Bowen et al., 2000).
According to the previous authors, the colon is made up of the following parts (figure: 1):
● The ascending colon , which located on the right side of the abdomen, is about 12.5 cm long. It is the part of the colon from the cecum to the hepatic flexure (the turn of the colon by the liver). It is retroperitoneal in most humans.
● The transverse colon which is the part of the colon from the hepatic flexure (the turn of the colon by the liver) to the splenic flexure (the turn of the colon by the spleen). The transverse colon hangs off the stomach, attached to it by a wide band of tissue called the greater omentum. On the posterior side, the transverse colon is connected to the posterior abdominal wall by a mesentery known as the transverse mesocolon. The transverse colon is encased in peritoneum and is therefore mobile (unlike the parts of the colon immediately before and after it). More cancers form as the large intestine goes along and the contents become more solid (water is removed) in order to form feces.
• The descending colon which is the part of the colon from the splenic flexure to the beginning of the sigmoid colon. It is retroperitoneal in two thirds of humans. In the other third, it has a usually short mesentery. 11
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● The sigmoid colon which is the part of the large intestine after the descending colon and before the rectum. The name sigmoid means S shaped. The walls of the sigmoid colon are muscular and contract to increase the pressure inside the colon, causing the stool to move into the rectum. Due to the intermittent high pressure within it, the colon can develop pockets called diverticuli in its walls. The presence of diverticuli, whether harmful or not, is called diverticulosis. Infection of the diverticuli is called diverticulitis.
Arteries provide blood to the colon and veins take the blood back toward the heart. The excess fluid drained to the bloodstream through a network of vessels called lymphatic tissue and lymph nodes. Lymph nodes also help fight infections (Rubin, 2001).
Figure (1): colon anatomy
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(2) Colorectal lesions:
Colorectal lesions can be divided into two main categories; The first , Inflammatory Bowel Disease ( IBD) which is a term that describes two diseases : Crohn's disease and ulcerative colitis. Although these two disorders usually differ enough to be distinguishable, they have certain common features (Baumgart and Sandborn 2007).
1 Crohn 's disease is a transmural, chronic inflammatory disease that may affect any part of the digestive tract but occurs principally in the distal small intestine and occasionally the right colon. It may be referred terminal ileitis and regional ileitis when it involves mainly the ileum and granulomatous colitis and transmural colitis when it principally affects the colon (Baumgart and Sandborn 2007).
2 Ulcerative Colitis is a chronic superficial inflammation of the colon and rectum, which characterized by chronic diarrhea and rectal bleeding, with a pattern of exacerbation and remissions and with possibility of serious local and systemic complications. The disorder occurs principally, but not exclusively, in young adults. The cause of ulcerative colitis is not known. Attempts to implicate a viral or bacterial agent have given only inconsistent results. Three major pathological features characterized ulcerative colitis and help to differentiate it from other inflammatory conditions:
a Ulcerative colitis is a diffuse disease.
It usually extends from the most distal part of the rectum for a variable distance proximally. When the disease involves the rectum alone, it is referred to as ulcerative proctitis . When the inflammatory process extends toward the splenic flexure, the terms proctosigmoiditis and left sided colitis are applied. Sparing of the rectum or involvement of the right side of the colon alone is rare and suggests the possibility of another disorder, such as Crohn disease (Askling et al 2001).
b The inflammatory process of ulcerative colitis is predominantly limited to the colon and rectum. It rarely involves the small intestine, stomach, or esophagus. When the cecum is affected, the disease ends at the
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ile ocecal valve, although minor inflammation of the adjacent ileum is sometimes noted (Rhodes, 1996).
c Ulcerative colitis is essentially a disease of the mucosa. Involvement of deeper layers is uncommon, occurring only in fulminate cases, usually in association with toxic megacolon (Baumgart and Sandborn 2007).
Morphological sequence of ulcerative colitis may develop rapidly or over a course of years as the following;
*Early colitis: Early in the evolution of the disease, in which the mucosal surface appears raw, red, granular and bleeds easily. *progressive colitis : As the disease continues, mucosal folds are lost. Lateral extension of crypt abscesses can undermine the mucosa, leaving areas of ulceration adjacent to hanging fragments of mucosa. *Advanced colitis: In long standing cases, the large bowel is often shortened, especially in the left side. The mucosal folds are indistinct and are replaced by a granular or smooth mucosal pattern. (Baumgart and Sandborn 2007)
The ulcerative colitis can be classified clinically, into three types:
*Mild colitis: Half of patient with UC have mild disease. Their major symptom is rectal bleeding. The disease in these patients is usually limited to the rectum but extend to the distal sigmoid colon. *Moderate colitis: The patients usually have recurrent episodes of loose bloody stools, crampy abdominal pain and frequently low grade fever. *Severe colitis: The patients have more than 6, and sometimes more than 20, bloody bowel movements daily, and about 15% of the patients with fulminant UC die of the disease. (Baumgart and Sandborn 2007)
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The second, Polyps of the colon and rectum (polyposis)
A gastrointestinal polyp is defined as a mass that protrudes into the lumen of the gut (Figure: 3). Polyps are subdivided according to their attachment to the bowel wall (e.g., sessile or pedunculated, with descrete stalk), their histopathological appearance (e.g., hyperplastic, serrated or adenomatous) and their neoplastic potential (i.e., benign or malignant). By themselves, polyps are only infrequently symptomatic and their clinical importance lies in their potential for malignant transformation (Figure: 2).
1 Adenomatous polyps (tubular adenomas) are pre malignant lesions. They are neoplasms that arise from the mucosal epithelium and composed of neoplastic epithelial cells that have migrated to the surface and have accumulated beyond the needs for replacement of the cells sloughed into the lumen. Adenomatous polyps can be classified into tubular, villous and tubulo villous types. The origin of colon cancer from adenomatous polyps is supported by the following: * The average age at onset of adenomatous polyps is earlier than that of colorectal cancer, suggesting that the later follows the former. Adenomatous polyps tend to antedate colon cancer by 10 to15 years. * Carcinomas are found in adenomas and some carcinomas have adenomatous remnants at their periphery. * In geographical regions in which there is a high risk of colorectal cancer, adenomatous polyps tend to be larger, are more often villous, and display higher grade dysplasia than those in low risk areas. The anatomical distribution of adenomas and carcinomas is similar, both being most frequent in the sigmoid colon in Western countries ( Rubin, 2001).
2 Familial Adenomatous Polyposis (FAP) , also termed adenomatous polyposis coli (APC), a rare, autosomal dominant inherited trait with almost complete penetrance, accounts for less than 1 percent of colorectal cancers. It is characterized by the progressive development of innumerable adenomatous polyps of the colorectum, particularly in the rectosigmoid region (Rubin, 2001).
3 Hyperplastic polyps (Metaplastic polyps) are small, sessile mucosal excrescences that display exaggerated crypt architecture. They are the most common polypoid lesions of the colon and are particularly frequent in the rectum. Hyperplastic polyps present in 40 15
Review of literature percent of rectal specimens in persons younger than 40 years and in 75 percent of older persons. These polyps are more common than usual in colons that contain adenomatous polyps and in populations with higher rates of colorectal cancer. Thus, these asymptomatic lesions reflect an increased risk of colorectal cancer (Rubin, 2001).
4 Juvenile polyps (Retention polyps) which are classified as hamaratomtous proliferations of the colonic mucosa. They are most common in children younger than 10 years, although one third occur in adults. They are single or multiple and occur most commonly in the rectum, although they may be seen anywhere in the small or large bowel (Rubin, 2001).
Figure (2): Diagram shows the progress of Colon cancer start from colon polyps.
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(3) Colorectal cancer :
It is the malignant cells found in the colon or rectum. The colon and the rectum are part of the large intestine, which is part of the digestive system. Because colon cancer and rectal cancers have many features in common, they are sometimes referred to together as colorectal cancer. Cancerous tumors found in the colon or rectum also may spread to other parts of the body (figure: 2 and 3) (American Cancer Society, 2002).
Figure (3): colorectal anatomy diagram show different lesions and cancer anatomy.
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{(a) Colorectal cancer progress:
Once cancer has been diagnosed, health care professionals use a process called "staging" to characterize the cancer based on whether the disease has spread to other parts of the body, and if so, to define how far the cancer has advanced. In colorectal cancer, there are five stages (Figure: 4).
* Stage 0: Called carcinoma in situ, this stage of colorectal cancer is confined to the innermost lining of the colon or rectum only. Surgery is the standard treatment and the cure rate is high.
* Stage I: Cancer has spread to the second and third layers of the lining and penetrated the inside wall of the colon or rectum, but has not spread to the outer wall or outside of the colon or rectum. This stage is also called "Dukes′ A" colon or rectal cancer. In this stage, patients may hear their doctors say the cancer is localized. Surgery is the standard treatment, and the cure rate is high. . . *Stage II: Cancer has spread outside the colon or rectum to nearby tissue, but has not invaded the lymph nodes. This is sometimes called "Dukes′ B" colon or rectal cancer. Surgery is standard treatment, although in some individual patient chemotherapy, radiation therapy, or a combination of the two may be appropriate. There are also clinical trials in which researchers are studying various treatment regimens chemotherapy, radiation therapy, or biologic therapy alone or in combination.
*Stage III: Cancer has advanced into nearby lymph nodes, but has not spread to other parts of the body. Sometimes this is called "Dukes′ C" colon or rectal cancer. The standard treatment is surgery followed by six months of chemotherapy. In some individual patients, radiation therapy may be appropriate. There are also clinical trials in which researchers are studying various treatment regimens chemotherapy, radiation therapy, or biological therapies, alone or in combination.
*Stage IV: Cancer has spread to other parts of the body. This stage is also called "Dukes′ D" colon or rectal cancer. Treatment options are complex, but most likely will include surgery and chemotherapy,
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The outlook for people with colorectal cancer is closely related to the stage at which their cancer is discovered and treated. According to the American Cancer Society, 37 percent of colorectal cancers are found before the cancer has spread and 90 percent of people whose cancer is found at this time will live at least five years, a time period commonly used to measure cancer survival rates. After colorectal cancer has spread to lymph nodes or other organs, the five year survival rate is 65 percent. The rate for people with advanced cases, where the disease has spread to distant parts of the body, is 9 percent (American cancer society, Inc, 2002). .
Figure (4): schematic diagram for different colorectal cancer stages.
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(b) The symptoms of colorectal cancer
The following are the most common symptoms of colorectal cancer. However, each individual may experience symptoms differently.
People who have any of the following symptoms should check with their physicians, especially if they are over 40 years old or have a personal or family history of the disease: a change in bowel habits such as diarrhea, constipation or narrowing of the stool that lasts for more than a few days
• rectal bleeding or blood in the stool • cramping or gnawing stomach pain • decreased appetite • vomiting • weakness and fatigue • jaundice yellowing of the skin and eyes
The symptoms of colorectal cancer may resemble other conditions, such as infections, hemorrhoids and inflammatory bowel disease. It is also possible to have colon cancer and not have any symptoms. (American cancer society, Inc, 2002)
(c) Risk factors of colorectal cancer:
It is anything that may increase a person's chance of developing a disease, such as smoking, diet, family history, or many other things. Different diseases, including cancers, have different risk factors, although these factors can increase a person's risk, they do not necessarily cause the disease. Some people with one or more risk factors never develop the disease, while others develop disease and have no known risk factors. But, knowing your risk factors to any disease can help to guide you into the appropriate actions, including changing behaviors and being clinically monitored for the disease (American cancer society, Inc, 2002).
The patients of colon cancer can be classified according to their risk factors into:
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1 Average Risk Those considered to be at average risk are those who are symptom free and have no personal or family medical history characteristics that would categorize their risk as moderate or high. Any one with symptoms should undergo a diagnostic work up. Methods should be selected based on consideration of community screening resources and quality and the patient’s medical status. When one test fails to provide sufficient information, another test should serve as a supplement. Although digital rectal examinations are no longer a part of the annual recommendations for screening asymptomatic adults, they are assumed to be a routine element of sigmoidoscopy (American Cancer Society, 2000).
2 Moderate Risk Risk for colorectal cancer is higher in those who are symptoms free but have a history of Adenomatous polyps or of cancer, including ovarian, uterine, or colorectal cancer. Screening in these individuals and those at high risk becomes more aggressive: it may be initiated earlier, be performed more frequently, or become more intensive by the use of more sensitive methods (American Cancer Society, 2000).
3 High Risk Higher risk requires higher level testing and associated intervention, making those methods capable of adenoma removal— colonoscopy and flexible sigmoidoscopy which are the preferred evaluations for patients with a personal history of inflammatory bowel disease or a family history of familial adenomatous polyposis or HNPCC. Patients with inflammatory bowel disease can expect colorectal cancer that does develop to be equal to their bowel disease in duration and extent. Screening begins with colonoscopy and biopsy of random sites in the colon. Despite the absence of direct evidence of the effectiveness of this approach, the rationale informing it is that early detection of dysplasia would prompt management likely to lower the risk of invasive carcinoma (American Cancer Society, 2000).
(d) Methods of colorectal cancer screening
Health care providers may suggest one or more of the following tests for colorectal cancer screening:
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1 Fecal occult blood test (FOBT) : This test checks for hidden blood in fecal material (stool). Currently, two types of FOBT are available. One type, called guaiac FOBT, uses the chemical guaiac to detect heme in stool. Heme is the iron containing component of the blood protein hemoglobin. The other type of FOBT, called immunochemical FOBT, uses antibodies to detect human hemoglobin protein in stool. Studies have shown that FOBT, when performed every 1 to 2 years in people ages 50 to 80, can help reduce the number of deaths due to colorectal cancer by 15 to 33 percent (Ouyang et al., 2005).
2 Sigmoidoscopy : In this test, the rectum and lower colon are examined using a lighted instrument called a sigmoidoscope. During sigmoidoscopy, precancerous and cancerous growths in the rectum and lower colon can be found and either removed or biopsied. Studies suggest that regular screening with sigmoidoscopy after age 50 can help reduce the number of deaths from colorectal cancer. A thorough cleansing of the lower colon is necessary for this test (Ouyang et al., 2005).
3 Colonoscopy : In this test, the rectum and entire colon are examined using a lighted instrument called a colonoscope. During colonoscopy, precancerous and cancerous growths throughout the colon can be found and either removed or biopsied, including growths in the upper part of the colon, where they would be missed by sigmoidoscopy. However, it is not yet known for certain whether colonoscopy can help reduce the number of deaths from colorectal cancer. A thorough cleansing of the colon is necessary before this test, and most patients receive some form of sedation (Ouyang et al., 2005).
4 Virtual colonoscopy (also called computerized tomographic colonography) : In this test, special x ray equipment is used to produce pictures of the colon and rectum. A computer then assembles these pictures into detailed images that can show polyps and other abnormalities. Because it is less invasive than standard colonoscopy and sedation is not needed, virtual colonoscopy may cause less discomfort and take less time to perform. As with standard colonoscopy, a thorough cleansing of the colon is necessary before this test. Whether virtual colonoscopy can reduce the number of deaths from colorectal cancer is not yet known (Winawer et al., 1997). 22
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5 Double contrast barium enema (DCBE) : In this test, a series of x rays of the entire colon and rectum are taken after the patient is given an enema with a barium solution and air is introduced into the colon. The barium and air help to outline the colon and rectum on the x rays. Research shows that DCBE may miss small polyps. It detects about 30 to 50 percent of the cancers that can be found with standard colonoscopy (Winawer et al., 1997).
6 Digital rectal exam (DRE) : In this test, a health care provider inserts a lubricated, gloved finger into the rectum to feel for abnormal areas. DRE allows examination of only the lower part of the rectum. It is often performed as part of a routine physical examination (Ouyang et al., 2005).
7 Molecular markers:
Biomarkers are defined as cellular, biochemical, molecular or genetic alterations by which a normal, abnormal or simply biologic process can be recognized or monitored. Biomarkers are measurable in biological media, such as human tissues, cells or fluids. Biomarkers could be used to identify pathological processes before individuals become symptomatic or to identify individuals who are susceptible to cancer ( Srinivas et al., 2001). To be useful clinically, tests for biomarkers must have high predictive accuracy and be easily measurable and reproducible, minimally invasive and acceptable to patients and physicians. Potential uses of biomarkers include the following: 1) monitoring patients with established cancer for recurrence, 2) early detection of asymptomatic patients, 3) aiding in the diagnosis of symptomatic patients, 4) surveillance of individuals known to be at high risk of cancer, and 5) surrogate endpoint markers for primary prevention strategies such as chemoprevention (Srivastava and Gopal Srivastava , 2002).
Recent advances in fundamental and clinical cancer research have mostly focused on identifying mutations, translocations and other molecular genetic abnormalities in the DNA of patients with established cancers. Such work is of great importance in the delineation of the mechanisms of tumor induction and for the identification of individuals who have an inherited predisposition to cancer. However, if early detection is to be useful, then clearly it must be targeted toward detection of lesions early enough to intervene and change the outcome. Although the existence of 23
Review of literature mutations in some known oncogenes is quite high in some specific cancers, in others it is insufficient to be of benefit in routine clinical practice. On the contrary, RNA and protein based diagnostic techniques may have distinct advantages over DNA based techniques in that they give direct evidence of abnormal gene expression at the time of sampling in a given patient who may not be symptomatic. Therefore, both DNA and protein based techniques are complementary. Identification of DNA sequences that encode proteins can stimulate subsequent expression analysis in tissues or biological fluids (Verma et al., 2001 and Smith et al., 2004).
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(4)Epidemiology of colorectal lesions and cancer
● Epidemiology of colorectal lesions:
Crohn’s disease occurs throughout the world, with an annual incidence of 0 5 to 5 per 100,000. Reports from various countries indicate that the incidence has increased dramatically over the past 30 years. The disease usually appears in adolescents or young adults and is most common among persons of European origin. There is a slight female predominance (1 6:1) (Rubin, 2001).
Ulcerative Colitis has an annual incidence of 4 to 7 per 100,000 populations and a prevalence of 40 to 80 per 100,000 in Europe and North America. The disease usually begins in early adult life, with a peak incidence in the third decade of life. However, it also occurs in childhood and in old age. In the United States, whites are affected more commonly than blacks (Benhattar, and Saraga, 1995 and Riegler et al., 1998).
Polyposis: The prevalence of adenomatous polyps of the colon is highest in industrialized countries. The diet is the only consistent environmental difference between high risk and low risk populations that has been identified. There is a modest male predominance (1.4:1) and blacks have a higher proportion of right sided adenomas and cancers (American Cancer Society, 2002).
● Epidemiology of colorectal cancer:
Colorectal cancer is the third most common cancer in both men and women. It is estimated by the American Cancer Society that 106,370 colon and 40,570 rectal cancer cases are reported in 2004. However, the number of new cases of colorectal cancer and the number of deaths due to colorectal cancer, have decreased, which is attributed to increased screening and polyp removal (American Cancer Society, 2002).
Colon cancer shares many environmental risk factors and both are found in individuals with specific genetic syndrome; however, there are some differences (WHO, 1997). Incidence rates vary approximately 20 fold around the world, with the highest rates seen in the developed world and the lowest in India (Muir et al., 1987 and Parkin et al., 1992). The 20 fold international difference 25
Review of literature may be explained by dietary and other environmental differences (Parkin et al., 1992), thus colorectal cancer is related to both genes and environment (Potter, 1999).
Colon cancer is the only cancer that occurs with approximately equal frequency in men and women (McMichael and Potter, 1980). However, in high incidence areas such as North America and Australia, as well as in Japan and Italy where rates are rising rapidly, rates in men now exceed those in women by as much as 20%. Rectal cancer is up to twice as common in men as in women (Ries et al., 1990).
Types of colorectal cancer:
Colorectal cancers have been classified as sporadic or hereditary (Goldberg and Winawe, 1993).
I Sporadic colorectal cancer which comprises about 60% of all colorectal malignancies (Souza, 2001). In last decades it has been confirmed that these sporadic forms originate from colorectal adenoma, through adenoma carcinoma sequence (Fearon and Vogelstein, 1990). This classic multi step model process occurs over a period of approximately 10 years (Muto et al., 1975).
II Hereditary colorectal cancer which is divided into two groups according to molecular genetic findings: (1) Tumors which show microsatellite instability, occur more frequently in the right colon, have diploid DNA and harbor characteristic mutations (e.g. transforming growth factor type II receptor and BAX (hereditary nonpolyposis colorectal cancer, HNPCC gene). (2) Tumors with chromosomal instability, which tend to be left sided, show aneuploid DNA, harbor characteristic mutations such as K ras, APC and p53 (familial adenomatous polyposis, FAP) (Lynch and delaChapelle, 1999). Hereditary CRCs are detected in several hamartomatous polyposis syndromes as juvenile polyposis (Bond, 1998).
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(5) Colorectal carcinoma in Egyptian patients
Colorectal cancer is the fourth common cancer and the second most common causes of cancer deaths in United States where the vast majority of cases occurs over age 60years (Greenle et al., 2000). In contrast, this disease is uncommon in developing countries (Magrath and Litvak, 1993), including Egypt where it is only the fifth most common cause of cancer deaths (Soliman et al., 1999). However, the percent of young onset colorectal cancer cases in Egyptians is strikingly high with more than one third of cases occurring under age of 40 years and the age adjusted mortality rates in young Egyptians are likewise high (Soliman et al., 1997;1999). In addition, rectal cancer is frequently high (Soliman et al., 1999).
The molecular pathology of colorectal carcinoma in Egypt differs from Western patients and between younger and older Egyptians (Soliman et al., 2001). Although Arabic countries share some cultural background and environmental exposures (Temtamy et al., 1994 and Al Saleh et al., 1998), rates of young onset disease are higher in Egypt than in Jordan (Al Jaberi et al., 1997), Lebanon (Adib et al., 1998), Algeria (Parkin et al., 1992) and Saudi Arabia (Isbister,1992). The rectal predominance in Egyptian cases occurred in both younger and older patients and was unrelated to urban\rural residence, which associated with very different environmental exposure (Soliman et al., 2001). Westernization of Egypt is occurring as the country develops and is affecting young people first because they are likely to change lifestyle than older people. The remarkable differences in molecular pathology as compared to western patients could result from inherent differences in sensitivity and molecular responses to western lifestyle in Egyptians (Rockhill and Giovannicci, 1999).
The studies on colorectal cancers in Egypt showed a high proportion of cases occurring before age 40 years. Unusual pathologic features in this population include few K ras mutations (11 %), high frequency of rectal tumors (51 %), and poorly differentiated histology (58%) and high levels of micro satellite instability (MSI H, 37%). (Chan, On on, Annie., 2002). Studying of unusual cancer distribution and analysis of particular genetic mutations may provide clues to cancer etiology (Soliman et al., 2001)
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(6) Molecular pathways of colorectal cancer
The evidence suggests that there are at least four pathways from normal cell to colorectal cancer.
1 The adenoma carcinoma sequence: Colorectal carcinoma arises through a series of well characterize histopathological changes as a result of specific genetic hits in a handful of oncogenes and tumor suppressor genes. Fearon and Vogelstein, 1990 reported that the analysis of genetic alterations of the tumor suppressor genes and oncogenes at different stages of the adenoma carcinoma sequence has allowed to develop a model for the clonal evolution of colorectal tumors by acquisition of sequential mutations (figure:5). At least, four sequential genetic changes need to occur to ensure colorectal evolution. One oncogene (K ras) and three tumor suppressor genes (APC, SMAD4 and TP53) are the main targets of these genetic changes. The dominant or recessive nature of these genes predicts that at least seven mutations are required: one oncogenic mutation at K ras and six additional ones to inactivate both alleles of the tumor suppressor genes (Kinzler and Vogelstein, 1996; and Kinzler and Vogelstein, 1998).
2 Loss of DNA mismatch repair (MMR) function: It is possible to lose DNA MMR function not by somatic mutation but by hypermethylation. Hypermethylation of hMLH1 now appears to be a very early event in many sporadic colorectal tumors (Kane et al., 1997), an epigenetic event that leads to a mutator phenotype. There is a rapid switch of methylation pattern affecting whole regions of the genome (Ahuja et al., 1997 and Myohanen et al., 1998).
3 Ulcerative colitis pathway: Several studies have indicated that patients with ulcerative colitis have a 2 8.2 relative risk of colorectal cancer compared with the normal population, accounting for about 2% of colorectal cancer. Loss of heterozygosity occurs colonally in both the adenoma carcinoma sequence and ulcerative colitis associated neoplasia. Many of these loci are already associated with one or more known candidate tumor suppressor genes these include 3p21 (B catenin gene), 5q21 (APC gene), 9p (P16 and P15 genes), 28
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13q (retinoblastoma gene), 17p (P53), 17q (BRCA1 gene), 18q (DCC and SMAD4 genes) and less frequently 16q (E cadherin gene) (Hardy et al., 2000).
4 Silencing of estrogene receptor (ER): There is evidence to show that almost all colon cancers arise from cells in which the ER gene has been silenced (Issa et al., 1994). Hypermethylation of ER is an age related phenomenon. There are also other genes that show hypermethylation with age. It is not known whether these early crucial step; moreover, it is not established why loss of the ER protein is so critical to colonic epithelial (Potter, 1999).
Figure (5): Proposed adenoma to carcinoma sequence in colorectal cancer.Adenomatous polyposis coli (APC) gene mutations and hypermethylation occur early, followed by k ras mutations. Deleted in colon cancer (DCC) and p53 gene mutations occur later in the sequence, although the exact order may vary.
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III- GenGeneeeessss involved in the pathways of colorectal cancer
Two classes of genes have been shown to drive the process of colonic neoplasia, "Gatekeepers" and "Caretakers". Gatekeepers are the genes that directly control cell birth and cell death. In the normal colon, the rate of cell birth precisely equals that of cell death, resulting in a constant number of colon cells. When colon gatekeeper genes are altered through mutation, the rate of cell birth exceeds that of cell death and a tumor is initiated. These tumors progress, becoming larger, more disorganized, and more dangerous, as mutations in gatekeeping genes accumulate during the tumorigenic process (Vogelstein et al., 1988 and Vogelstein et al., 2000). Caretakers, the genes of second class, do not directly control cell birth or cell death but rather control the rate of mutations of other genes, including gatekeeper genes. When caretakers are altered, the cell accumulates mutations at a high rate and the process of tumorigenesis is accelerated (Vogelstein et al., 1988 and Vogelstein et al., 2000). . A Gatekeeper of colorectal cancer:
The APC gene is the major gatekeeper of the colon. Patients with inherited mutations of this gene develop thousands of benign tumors of the colon, called adenomas. Vogelstein group has helped to discover the mechanisms through which APC regulates colon cell growth. APC binds to another protein, β catenin and stimulates its phosphorylation. In cells with an APC mutation, this phosphorylation does not take place and β catenin is activated. The intracellular location of mutant β catenin changes upon mutation; it migrates to the nucleus, interacts with transcription factors and induces the expression of genes that stimulate cell birth (Vogelstein et al., 1988 and Vogelstein et al., 2000).
Mutations in APC or β catenin are sufficient to initiate the growth of a small benign tumor but are not sufficient to make such tumors progress to more advanced forms. Several other pathways participate in such progression. One of these pathways involves transforming growth factors β (TGF β), a small polypeptide hormone that negatively controls colon cell growth. Vogelstein group has shown that the genes responsible for mediating the 30
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effects of TGF β are often disrupted in colorectal tumors (Vogelstein et al., 1988 and Vogelstein et al., 2000).
A second critical pathway involved in tumor progression involves p53, a gene that is inactivated not only in colorectal cancers but also in most other cancers types. Activation of the normal p53 gene inhibits cell growth by blocking the cell cycle or by stimulating cellular suicide (apoptosis). Vogelstein group previously discovered two of the genes that mediate the effects of p53 on the cell cycle. Vogelstein group had discovered a gene, called PUMA (for p53 upregulated mediator of apoptosis), that appears to be a key mediator of the apoptotic function of p53. The PUMA protein is located in mitochondria and binds to other, homologous proteins known to be involved in cell death. Cell in which the PUMA gene is purposefully disrupted through genetic targeting are resistant to certain forms of apoptosis (Vogelstein et al., 1988 and Vogelstein et al., 2000).
B Caretakers in colorectal cancer:
One caretaker system that functions to limit colorectal neoplasia involves mismatch repair. This system repairs sutable mistakes made during copying of the genome by DNA polymerases. Vogelstein group, in conjunction with that of Albert et a l (1994) and others, have shown that inherited alterations of these genes lead to hereditary nonpolyposis colorectal cancer (HNPCC), the most common hereditary cancer now known (Vogelstein et al., 1988 and Vogelstein et al., 2000).
Colorectal cancers that do not occur in a heritable form generally do not have defects in mismatch repair but rather have a different form of caretaker defect, named CIN, for chromosome instability. CIN tumors develop lose or gain of large parts of chromosomes at a rate that is much higher than that of normal cells. Vogelstein group has recently shown that CIN develops very early during the colon tumorigenic process. Experiments to identify the responsible genes are in progress (Vogelstein et al., 1988 and Vogelstein et al., 2000).
There were certain stabilized genes involving in colon cancer developing (Figure 6). These genes can be summarized as follow: 31
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(1) APC gene
The Adenomatous polyposis coli (APC) gene can be regarded as the gene of colorectal cancer. Germ line mutations in the APC gene result in familial Adenomatous polyposis (FAP) (Kinzler and Vogelstein, 1996), one of the principle hereditary predisposition to colorectal cancer. Somatic APC mutations are also found in the most sporadic tumors (Powell et al., 1992).
The main tumor suppressing function of APC resides in its capacity to regulate intracellular ß catenin levels (Korinek et al., 1997). Most APC mutations results in truncated proteins that lack all axin\conductin binding motifs and variable numbers of the 20 amino acid repeats that are associated with the down regulation of intracellular ß catenin levels (Miyaki et al., 1994).
(2) P53 gene:
The tumor suppressor protein p53 is a multifunctional transcription factor involved in the control of cell cycle progression, DNA integrity and cell survival. P53 gene is mutated in half of all tumors and has a wide spectrum of mutation types. The gene (p53) mutants show different degrees of dominance over coexpressed wild type p53 and loss of the wild type p53 allele has been observed frequently. Several p53 mutants can exert oncogenic functions beyond their negative domination over the wild type p53 tumor suppressor functions. These so called gain of function effects, such as enhancement of tumorigenicity and therapy resistance, were investigated in p53 null cells. The possible mechanisms by which p53 mutants exert their gain function effects are reviewed. The existence of functional gains of certain p53 mutants has important ramifications for tumor prognosis and cancer therapies (van Oijen and Slootweg, 2000).
TP53 mutation in colorectal cancer: Mutations of p53 are found in approximately half of all colorectal cancer cases, with a higher frequency observed in distal colon and rectal tumors and a lower frequency in proximal, mucinous, and MSI tumors. There are some evidences that different p53 mutations are associated with different clinical properties including prognosis and response to therapy, although further large 32
Review of literature studies are required to establish this. P53 status appears to have predictive value for the survival benefit of colorectal cancer patients from 5 Fluro Uracil chemotherapy (Iacopetta, 2003).
Mutations in five hotspot codons (175, 245, 248, 273, and 282) account for approximately 43.5% of all p53 mutations in colorectal cancer ( Soong et al., 2000; Soussi et al., 2000; Soussi and Beroud, 2003). Interestingly, mutations occurring in the conserved regions of p53 are more frequent in tumors from the distal compared to proximal colon and this has been suggested to reflect a different etiology (Jernvall et al., 1997). Transversion rather than transition mutations are also reported to occur more frequently in distal tumors, again possibly reflecting different etiology between right and left sided colorectal cancer (Borresen et al., 1998).
Both mutation and over expression occur more frequently in distal compared to proximal tumors by a factor likely to be in the range of 1.5 3 folds. P53 alterations are also more frequent in tumors that are aneuploid, non mucinous and do not show either the microsatellite instability (MSI ) or methylator (CIMP ) molecular phenotype (Iacopetta, 2003).
(3) K ras gene
The ras genes were the first oncogenes to be implicated in human cancer. The experiments to isolate and characterize ras transforming genes were in many ways landmarks of molecular oncology. The ras oncogenes were initially discovered as the transforming genes of the Harvey and Kirsten murine sarcoma viruses (Ha MSV, Ki MSV) (Harvey, 1964 and Kirsten & Mayer, 1967).
The ras proteins have provided in the last 15 years a workhorse for normal and tumorigenic cellular signal transduction. These proteins are not only involved in a high percentage of human tumors but also are a central point for many signal transduction pathways in the cell. The analysis of these molecules had led to the understanding of the relationship between tumor development and signal transduction (Malumbres and Pellicer, 1998).
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Three genes encode Ras proteins: N ras, H ras, and K ras. The three Ras proteins produced from these genes are closely related to one another and are similar in their ability to interact with regulators. The distribution of ras gene mutations does show some tissue specificity (Adjel, 2001).
The K ras gene has constitutive GTPase activity, which is lost when the gene is mutated, most commonly at codon 12, 13, and 61. Mutation in K ras lead to increased and unregulated cellular proliferation and malignant transformation ( Bos, 1989).
In colorectal cancer cases the majority of K ras mutations were found in codon 12, with a smaller number of nucleotide substitutions in codon 13. K ras mutations were significantly more common in rectal (39%) than in colon tumors (20%), suggesting different etiologies for these cancers. The majority of K ras mutations were base pair transitions, occurring predominantly at the second bases of codons 12 and 13 (Andreyev et al., 1998).
Previous studies have reported K ras mutation rates to be higher in left sided colorectal cancers (Toribara and Sleisenger, 1995). Some studies reporting K ras mutation frequencies that are higher in patients with adenoma compared with colorectal cancer (Pretlow et al., 1993).
K ras mutations must also follow the loss of APC. If APC is intact, a gain of function K ras mutation appears to create only the microscopic lesion called an aberrant crypt focus, which is non neoplastic and is though to be non progressive. Invisible to colonoscopy, each is a site of thickened epithelium with an asteroid or oval shaped glandular lumen ( Andreyev et al., 1998).
(4) Deleted in colorectal cancer gene (DCC)
The DCC (deleted in colorectal cancer) gene was proposed as a putative tumor suppressor gene. Data supporting this proposal included observations that one DCC allele was deleted in roughly 70% of colorectal cancers, some cancers had somatic mutations of the DCC gene and DCC expression was often reduced or absent in colorectal cancer tissues and cell lines. Despite subsequent studies which have supported DCC’s potential role as a tumor suppressor
34
Review of literature gene, the rarity of point mutations identified in DCC coding sequences, the lack of a tumor predisposition phenotype in mice heterozygous for DCC inactivating mutations and the presence of other known and candidate tumor suppressor genes on chromosome 18q have raised questions about DCC’s candidacy. Following its initial characterization, the DCC protein was identified as a transmembrane receptor for netrins, key factors in axon guidance in the developing nervous system. At first glance, the established role of DCC and netrin 1 during organization of the spinal cord could be viewed as a further challenge to the position that DCC inactivation might play a significant role in tumorigenesis. However, many observations on DCC’s functions in intracellular signaling have renewed interest in the potential contribution of DCC inactivation to cancer. In particular, data indicate that, when engaged by netrin ligands, DCC may activate downstream signaling pathways. Moreover, in settings where netrin is absent or at low levels, DCC can promote apoptosis (Mehlen and Fearon, 2004).
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Figure (6): Schematic representation of the putative steps in colorectal cancer progression. Dysplastic aberrant crypt foci (ACF), early, intermediate or late adenoma، carcinoma and metastatic carcinoma are respectively the various steps of colorectal cancer progression. Adenomatous polyposis coli (APC) mutations are supposed to be preliminary events, while K RAS, DCC (deleted in colorectal cancer), SMADS, and p53 mutations appear as late events.
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IV --- KLF 666 tumor suppressor gene
(1) Introduction:
DNA binding transcriptional regulators are central to the control of the whole process (Mitchell and Tjian, 1989). The zinc finger is the most frequently represented DNA binding domain in transcription factors (Berg, 1990 ). The number, composition and organization of the zinc fingers segregate the corresponding proteins into evolutionarily related families (Klug and Schwabe, 1995). Amongst them is the mammalian family of C2H2 zinc finger Kruppel like factors (KLFs) (Turner and Crossley, 1999; Dang et al., 2000; Bieker, 2001).
Transcriptional factors are grouped into several classes, which include the helix loop helix, leucine zipper, homeodomain and zinc finger protein families. Some zinc finger transcriptional factors can be further subclassified based on their homology to the Drosophila Kruppel protein (Anderson et al., 1995).
The Kruppel gene of Drosophila is required for the correct formation of the embryonic body plan through its activity as a transcriptional repressor (Schuh et al., 1986; Stanojevic et al., 1989 and Licht et al., 1990). Members of the Kruppel like factor (KLF) gene family have been discovered in mammals; they are expressed in a wide variety of tissues and encode both transcriptional activator and repressor proteins (Turner and Crossley, 1999). In the study on targeted deletion in mice, it had been revealed the roles for members of the KLF family in erythroid cell maturation (Nuez et al., 1995 and Perkins et al., 1996), T cell activation (Kuo et al., 1997), and blood vessel stability (Kuo et al., 1997), as well as skin permeability (Segre et al., 1999). These results indicate that the mammalian KLF genes family plays an important role in the control of hematopoietic and other cell type differentiation (Oates et al., 2001).
The KLF proteins share a nearly identical carboxy terminal DNA binding domain, but have widely divergent amino terminal activation domains, patterns of expression and transcriptional targets (Bieker, 2001& Black et al., 2001). The KLF family is broadly involved in differentiation and development, growth related signal
37
Review of literature transduction, cell proliferation, apoptosis, and angiogenesis . These functions have drawn attention to their possible roles in tumorigenesis (Bieker, 2001& Black et al., 2001) .
Although the DNA binding activity of the highly conserved, C terminal zinc finger domain of KLF proteins is clear, the function of diverged, N terminal structures remains under investigation. Membership in the KLF family is defined by the presence of 3 tandem zinc finger motifs at the C terminus of the protein (Ruppert et al., 1988). This domain confers sequence specific DNA binding to GC rich elements of the general structure CCN CNC CCN (Klevit, 1991), such as the CACCC box (Miller and Bieker, 1993; Yet et al., 1998 and Van Vliet et al., 2000) and in vitro site selection has suggested the consensus sequence NNR CRC CYY (Shields and Yang, 1998). The N termini of KLF proteins are highly variable and several are characterized by the presence of domains with transcriptional activator and/or repressor functions in vitro (Bieker& Southwood, 1995; Turner&Crossley, 1998; Geiman et al., 2000 and Van Vliet et al., 2000), . In addition, the N terminus of some KLF proteins contains sites of phosphorylation and acetylation that may contribute to regulation of KLF activity ( Ouyang et al., 1998). Despite these studies, it remains an important task to identify functionally significant sites in KLF protein N termini (Bieker, 2001& Black et al., 2001).
KLFs are uniquely expressed in a large variety of tissues; bind to similar ‘‘GTbox or CACCC element’’ sites on DNA; and function as transcriptional activators, repressors or both (Turner and Crossley, 1999; Dang et al., 2000; Bieker, 2001). Fifteen KLF coding genes have been identified in the human and mouse genomes and segregated into four phylogenetically distinct groups based on structural considerations (Bieker, 2001).
The Kruppel like transcriptional factors so far cloned include erythroid Kruppel like factor (EKLF/KLF1), lung Kruppel like factor (LKLF/KLF2), basic Kruppel like factor (BKLF/KLF3), gut enriched Kruppel like factor (GKLF/KLF4), basic transcription factor element binding protein 2 (BTEB2/KLF5), Zf9/ CPBP/KLF6, ubiquitous Kruppel like factor (UKLF/KLF7), fetal erythroid Kruppel like factor (FKLF/KLF8) and basic transcription factor element binding protein (BTEB1/KLF9). The Zf9 gene was cloned
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Review of literature originally from cDNA libraries of activated rat stellate cells (Ratziu et al., 1998) and human placenta (Koritschoner et al., 1997).
A large body of work has demonstrated that KLFs regulate growth, proliferation and differentiation of distinct cell lineages by binding to similar DNA elements in the promoters of housekeeping and tissue specific genes (Turner and Crossley, 1999; Dang et al., 2000; Bieker, 2001). For example, targeted disruption of the mouse Klf1 gene leads to absence of adult b globin gene expression, changes in chromatin organization at the same locus, and uncoordinated erythroid cell proliferation (Nuez et al., 1995; Perkins et al., 1995; Coghill et al., 2001). Similarly, KLF4 has been shown to stimulate p21 accumulation and growth arrest in cell cultures, and to direct terminal differentiation of dermal and intestinal epithelia in mice (Shields et al., 1996; Segre et al., 1999 and Katz et al., 2002). Predominant expression of KLF5 in proliferating epithelial cells of the mouse intestine, on the other hand, has been associated with the ability of the transcription factor to confer serum, and anchorage independent growth to stably transfected cells (Sun et al., 2001; Dang et al., 2002). KLF like genes with comparable regulatory properties have been identified in lower vertebrates as well, thus documenting the ancient role of these nuclear proteins in regulating metazoan development (Huber et al., 2001;and Kawahara and Dawid, 2001).
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Review of literature
(2) KLF6 protein:
Members of the pregnancy specific glycoprotein (PSG) family and related genes do not have TATA boxes or the initiator in their promoter regions. To identify proteins that regulate PSG gene expression , Koritschoner et al. (1997) screened a placenta expression library with the core promoter element (CPE) from PSG5. They isolated cDNAs encoding a protein that they called 'core promoter element binding protein' (CBPB or COPEB). By sequence analysis, Koritschoner et al. (1997) found 2 possible translation initiation codons, use of which would produce predicted proteins of either 283 or 290 amino acids. COPEB contains an N terminal acidic domain, a serine/threonine rich central region, and 3 contiguous zinc finger domains in the C terminal region. The in vitro COPEB translation product had a mass of 32 kD by SDS PAGE. Using mammalian cells expressing COPEB, Koritschoner et al (1997) showed that COPEB is capable of activating transcription approximately 4 fold from the PSG5 promoter. A Gal4 COPEB fusion protein activated transcription from a heterologous promoter containing Gal4 binding sites. Northern blot analysis revealed that COPEB is expressed as a 4.5 kb mRNA in several tissues, with the highest levels in placenta.
KLF6 protein contains three Cys2 His2 zinc fingers in its carboxyl terminal domain and a unique amino terminal domain that is rich in serine/proline clusters (figure: 7), which could be potential targets for post translational modifications (Ratziu et al., 1998).
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Figure (7): KLF6 protein structure.
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The KLF6 sequence encodes a 283 amino acid protein with two distinct domains as shown in figure (8), a 201 aa proline and serine rich amino terminal activation domain and a carboxy terminal 82aa zinc finger C2H2 DNA binding domain. It is defined as a"Kruppel like factor" because this DNA binding domain (from amino acid 202 to aminoacid 283) shows homology to other members of the KLF family. In contrast, the KLF6 activation domain (from aminoacid 1 to aminoacid 201) is unique and only shares partial homology with another member of the KLF family, KLF7 (previously called' UKLF'). KLF6 has been established as a transcription factor that binds to promoter regions of genes containing a"GC Box"motif (Ratziu et al., 1998). KLF6 regulates the expression of a placental glycoprotein (Koritschoner et al.,1997), HIV 1 (Suzuki et al.,1998), collagen alfa l (Ratziu et al., 1998), TGFbl, types I and II TGFb receptors (Kim et al., 1998) and urokinase type plasminogen activator (uPA) (Kojima et al., 2000).
Figure (8): Functional domains of KLF6 protein
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(3) KLF6 gene nomenclature and identification:
The human Kruppel like transcription factor KLF6 was initially identified as the Core Promoter Binding Protein (CPBP), which was involved in the regulation of the Pregnancy Specific Glycoprotein genes (PSG) (Koritschoner et al., 1997). This factor, whose conceptual gene has been renamed in human and mouse as COPEB and more recently KLF6, localizes to human chromosome 10p15 as in Figure (9) (Onyango et al., 1998), a region which is deleted in various tumors. While the expression pattern and chromosomal location of KLF6 were known, the present invention provides the first evidence that KLF6 is inactivated in cancers and acts as a tumor suppressor gene (Ratziu et al., 1998).
Figure (9): Location of KLF6 gene on chromosome 10
The sequencing of human KLF6 gene revealed that, it consists of four exons and the three introns as diagrammed in figure (10) (Gehrau et al., 2005). The KLF6 mRNA is widely distributed in the organism, though is highly enriched in the placenta both at term and during embryogenesis (Koritschoner et al., 1997, Slavin et al., 1999 & Blanchon et al., 2001). KLFs recognize and bind GC rich DNA elements present in a variety of promoters of mammalian genes. For instance, these cis acting elements have been found to control certain genes involved in diverse biological functions such as cell cycle regulation and normal development (Black et al., 2001).
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Figure (10): Genomic structure of KLF6 gene. Exons are shown as black boxes and introns by lines in concordance with splicing process. 5\ and 3\ untranslated regions (UTR) are shown as gray boxes.
Transcriptional targets of KLF6 include the genes encoding PSG, TGFh and TGFh receptors type I and II, collagen alfa 1, the collagen specific molecular chaperone HSP47, leukotriene C(4) synthase; keratin 4 and 12, urokinase plasminogen activator, p21, endoglin, nitric oxide synthase and the insulin like growth factor I receptor (Okano et al., 2000; Zhao et al., 2000; Botella et al., 2002; Warke et al., 2003 and Rubinstein et al., 2004).
Kruppel like factor 6 (KLF6) is a ubiquitously expressed zinc finger transcription factor that identified as a tumor suppressor gene inactivated in sporadic prostate cancers. Distinct mutations localize to separate tumor foci within individual prostate tumors, indicating genetic heterogeneity with respect to KLF6 mutations. Wild type KLF6 up regulates the cell cycle inhibitor p21 in a TP53 independent manner and suppresses growth, whereas tumor derived KLF6 mutants fail to up regulate p21 or suppress proliferation prostatic and non small cancer cell. The high prevalence of KLF6 abnormalities in prostate cancer is particularly noteworthy, because mutations of other well characterized tumor suppressor genes such as TP53 occur infrequently in localized disease ( Narla et al., 2003; Chen et al., 2003 and Narla et al., 2005 ).
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(4) KLF 6 tumor suppressor gene and Cancer:
Kruppel like factor 6 (KLF6/Zf9/CPBP), a member of the Kruppel like family of zinc finger transcription factors, its function is broadly involved in differentiation and development, growth related signal transduction, cell proliferation, apoptosis and angiogenesis (Song et al., 2006 ). It has recently been suggested that it is a mutated tumor suppressor in several human cancers (Yin et al.,2007).
KLF6 is frequently inactivated in prostate and colorectal tumors (Narla et al., 2001 and Benzeno et al., 2004). Additionally, other reports suggested that KLF6 mutations are rare or infrequent in certain human populations (Chen et al., 2003; Koivisto et al., 2004 and Montanini et al., 2004). Other studies demonstrated that KLF6 is frequently down regulated in certain lung cancer derived cells and suppresses tumor growth via induction of apoptosis (Ito et al., 2004). Also in this context, it was shown that expression of KLF6 is attenuated in human glioblastoma when compared with primary astrocytes and that KLF6 might inhibit cellular transformation induced by several oncogenes (Kimmelman et al., 2004). Additionally, other report concluded that inactivating mutations of the KLF6 gene that occurred in certain gliomas might play a role in the pathogenesis of these tumors (Jeng and Hsu, 2003). Another study demonstrated that KLF6 plays a new role as an inhibitor of c Jun proto oncoprotein function via a proteasome dependent pathway (Salvin et al., 2004).
A number of reports suggest that diverse stimuli are important regulators of KLF6 gene expression. For example, it has been shown that KLF6 level is modulated by chemical and mechanical injury, hemorrhage, cytokines, tumor promoters and iono phores (Inuzuka et al., 1999; Kojima et al., 2000; Botella et al., 2002; and Kiang et al., 2004).
To gain further insight into the molecular mechanisms governing KLF6 expression, a human genomic sequence was isolated and analyzed to determine the structure of the KLF6 locus and the potential cis acting elements located at its 5\ flanking regulatory region. In addition, distinct KLF6 sequences derived from various species were subjected to computer aided alignments to search for evolutionary conserved clues. More importantly, the 45
Review of literature transcription start site of human KLF6 gene was determined and the activity of its promoter was investigated in different cell types (Gehrau et al., 2005).
Reeves et al (2004) reported that the pathogenic sequence of molecular changes in CRC is better established, which would allow to begin defining where abnormalities of KLF6 might occur within this temporal sequence. From the Reeves et al (2004) study, it was established that deregulation of KLF6 by a combination of allelic imbalance and mutation may play a role in the development of CRC.
KLF6 was inactivated by loss and/or mutation in most sporadic and IBD related CRCs. The KLF6 locus was deleted in at least 55% of tumors and mutations were identified in 44%. Rates of KLF6 loss and mutation were similar to those of TP53 and K RAS in the same samples. KLF6 mutations were present in tumors with either microsatellite or chromosomal instability and were more common, particularly in the IBD related cancers, in the presence of wild type APC. Unlike wild type KLF6, cancer derived KLF6 mutants neither suppressed growth nor induced p21 following transfection into cultured cells (Reeves et al., 2004).
Cho et al (2006) study revealed that five somatic mutations and a 42.9% allelic loss of the KLF6 gene in colorectal cancers were found. Thus genetic alteration of the KLF6 gene may play a role in the development of colorectal cancers.
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Subjects and Methods
Subjects and Methods
1- Samples collection:
The patients included for this study were 83 presented with different indications for colonoscopic examination. Colonoscopic examination was held in Kasr Al Eini Endoscopic Unit, Tropical Medicine Department, Faculty of Medicine, Cairo University. Selecting patients with colorectal cancer or colorectal pre cancerous lesions was done according to the results of tissue biopsy from lesion and adjacent normal.
This work is part of the ASTR project funded from Egyptian Academy of Science and Technology Research (project no. 103). Consent was obtained from the patients only after their physician surgeon has informed them of their diagnosis according to the institutional review board (IRB).
All laboratory work was carried out and standardized under blind conditions. For each patient part of the tissue sample was kept at 10% formalin for histopathology examination after E&H staining. The other part of sample was frozen and stored at 80˚C for DNA isolation.
Clinical data were collected from each patient including age, family history, histipathological grading , size, location, pathological stages, polyps size and presence of dysplasia. Moreover chief complaints leading to clinical presentation (i.e. obstruction, bloody stool, and change in bowel habit) were considered.
The patients were classified into three main groups;
I Cancerous group Thirty eight of patients were diagnosed as adenocarcinoma. The clinical data for those patients are shown in Table (1).
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Subjects and Methods
Table (1): The clinical data for the patients of adenocarcinoma group.
characters Number(%)
Age /year (mean±SD) =.44.9±11.7
Gender: Male …………………………………………… 16 (42) Female ……………………………………………. 22 (58)
Colonoscopic findings: Rectum ……………………………..……………….. 21(56) Sigmoid ……………………………………………… 5(13) Caecum ……………………………………………… 1(3) Hepatic ………………………………………………. 2(6) Ascending ………………………………………………. 4(11) Descending ………………………………..………………. 3 (8) Splenic ………………………………………………… 1(3) Missed ……………………………………………...….1(3 )
Symptoms: Bleeding ……………………...……………………… 25(71) Constipation ………………………………………………30(86) Diarrhea ………………………………………………..9(26) Weight loss …………………………………..……………28(80)
Histipathological grading: Grade (I) ……………………..………………………. . 3(8) Grade (II) ………………………………………………. 23(61) Grade(III) …………………….………………………. 3(8) Unknown …………………………………………………..9(23)
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Subjects and Methods
II polyps group : This group contained 23 patients which were diagnosed as adenomatous polyps (16 cases), familial adenomatous polyps (FAP) (3 cases) and Hyperplastic polyps (4 cases). The clinical data for those patients are shown in Table (2).
Table (2): The clinical data for the patients of polyps group (II)
characters Number(%)
Age /year (mean±SD) = 48±12.3
Gender: Male ………………………………………… 16(73) Female …………………………………………….. 6(27) Missed ………….………………………………….. 1
Colonoscopic findings: Rectum ……………………………………………….. 4(17) Sigmoid ………………………………………………... 8(35) Caecum ………………………………………………… 1(4) Hepatic ……………………………………………. 1(4) Ascending …….……………………………………………. 1(4) Descending ………………………………………………. 1 (4) Whole …………………………………………………….3(13) Transverse colon……………………………………….. ….3(13) Missed …………………………………………………… 1(4)
Symptoms Bleeding ………………………..………………………18(78) Constipation …………………………………………….10(43) Diarrhea ……………………………………………… 9 (39) Weight loss ……………………………………………… 1(4) Missed … …………………………………………… 3(13)
Polyps size ≤ 1cm ...... 19 (83) ≥ 1cm ...... 4 (17 )
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Subjects and Methods
III Inflammatory Bowel Diseases (IBD) Include 22 patients which were diagnosed as ulcerative colitis (19 cases) and Crohn's disease (CD) (3 cases). The clinical data for those patients are shown in Table (3).
Table (3): The clinical data for the patients of IBD group (III).
characters Number(%)
Age /year (mean±SD) = 43.3±15.6
Gender: Male ……………………………………………….. 16 (73) Female ……………………………………………….. 5(23) Missed ………………………………………… 1(4)
Colonoscopic findings: Rectum …………………………………………….. 1(4.5) Recto sigmoid …………………………………………4 (18) Left colon ……………………………………………....4(18) Up to Transverse colon… ……………………………….1(4.5) Whole ………………………………………………..5(23) Splenic ………………………………………………... 1(5) Missed ……………………………………………….6 (27)
Dysplasia (for U.C subgroup only) Present …….……..…………………………………6(32) Absent ……………..……..………………………..13(68 ) Symptoms Bleeding ………………………………………………17(77) Constipation ……………………….…………………….8 (36) Diarrhea ………………………………………………15 (68) Weight loss ………………………….…………………6(27) Missed ………………………………………………..1(5)
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Subjects and Methods
2- Genomic DNA extraction from tissue samples
The DNAs were extracted from the tissue samples using QIAamp ® DNA mini kit (QIAGEN, Germany) as follow;
1 25 mg of tissue were cut into small pieces and placed in 1.5ml microcentrifuge tube, 180ul of buffer ATL were added.
2 20 l of proteinase K were added then mixed by vortexing, and incubated at 56 oC until the tissue was completely lysed (over night). The tissue samples were placed on rocking platform during the incubation.
3 1.5 ml micocentrifuge tube was briefly centrifuged to remove drops from the inside of the lid.
4 200 l of buffer AL were added to the sample, mixed by pulse vortixing for 15 seconds, and incubated at 70 o C for 10 minutes then, 1.5ml micocentrifuge tube was centrifuged as above to remove drops from inside of the lid.
5 200 l ethanol (96 100%) were added to the sample and mixed by pulse vortexing for 15 seconds. After mixing, the 1.5ml micocentrifuge tube was briefly centrifuged to remove the drops from inside of the lid.
6 The mixture from step 5 was applied carefully to the QIAamp spin column without wetting the rim. The cap was closed and the tube was centrifuged at 8000 rpm for 1minute. The QIAamp spin column was placed in a clean 2ml collection tube and the tube which contains the filtrate has been discarded.
7 The QIAamp spin column has been opened carefully and 500ul buffer AW1 were added without wetting the rim. After closing the cap, the QIAamp spin column was centrifuged at 8000 rpm for 1 minute. Then it was placed in a clean 2ml collection tube and the collection tube containing the filtrate was discarded.
8 QIAamp spin column has been opened and 500 l buffer AW2 were added without wetting the rim. The cap was closed and the column was centrifuged at 14,000 rpm for 3 minutes.
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Subjects and Methods
9 The QIAamp spin column was placed in a clean 1.5ml micocentrifuge tube and the collection tube was discarded. Carefully, the QIAamp spin column was opened and 200ul of buffer AE was added. Then, it was incubated at room temperature for 5 minutes, after incubation, the column was centrifuged at 8000 rpm for 1 minute.
10 The step 9 was repeated and the DNA was obtained in the filtrate TE buffer.
11 The DNA was kept at 80 o C for PCR analysis.
3-Electrophoresis of extracted DNA
Genomic DNA isolated from tissue samples was electrophoresed using agarose gel electrophoresis to check successfulness of isolation and its purity.
Apparatus used:
1 Electrophoresis unit 8x11 cm (Bio RAD sub TM DNA cell). 2 Power supply (Hoefer Scientific Instruments). 3 Biodocument Analyzer (BioMetra, Germany).
Reagents:
1 EDTA 0.5 M, pH 8.0
EDTA (2H2O)……………………………………………..186.1g This weight was dissolved in about 500 ml ddH 2O and the pH was adjusted by pH meter using NaOH solution and then completes the volume to 1 liter with ddH 2O.
2 Tris acetate EDTA buffer (TAE) 50X Tris base…………………………………………………..242.0g Glacial acetic acid………………………………………..57.1 ml EDTA 0.5 M, pH 8.0……………………………………….100 ml These reagents were mixed thoroughly and the volume was completed to liter with ddH 2O. The buffer was stored at room temperature.
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Subjects and Methods
3 Tris Acetate EDTA buffer (TAE) 1X (working solution) A volume of 20 ml of stock 50X TAE buffer was added to volumetric cylinder and the volume was completed to 1 liter with ddH 2O.
4 Gel loading buffer Bromophenol blue (Bio RAD laboratories)……………. .0.025g Xylene Cyanol FF (Bio RAD laboratories)…………….. ..0.025g Glycerol……………………………………………………. 3 ml The reagents were mixed and the volume was completed to 10 ml with ddH2O and stored at 4ºC.
5 Agarose gel (1%): Agarose (Viogene)………………………………...... …….1g, TAE (1X)…………………………………………… …...100 ml Ethedium bromide …………………………………….....0.5 g/ml Agarose (1g) was added to 100ml TAE, cooked in a microwave for 4 minutes and left to cool to 60 o C. Three l of ethidium bromide were added. The gel was casted in the tray of a submarine and after drying it was immersed in the electrophoresis unit.
Procedure:
1 5 l of extracted DNA was mixed with 1 l loading buffer. 2 The samples were loaded on 1% agarose gel in running buffer TAE (1X) and electrophoresed at 25 volt for 2 hours. 3 The DNA was visualized under UV transilluminator using BioDocument Analyzer (Biometra, Germany) to indicate integrity, purity and the amount of extracted DNA. A photograph was taken using the same analyzer to have a permanent record.
4-Concentration detection of extracted DNA
A Principle
This method is based on the fact that the bases in the DNA molecule absorb ultraviolet (UV) light, with maximal absorbance at a wavelength of 260 nm. Because the concentration of base pairs available to absorb the UV light is a direct function of the concentration of the DNA itself, the A 260 (absorbance at 260 nm) 53
Subjects and Methods
can be used to measure the overall concentration of DNA in a solution.
B Procedure
1 5 l of extracted DNA were added to 45 l of ddH 2O. 2 Reading the absorbance of solution was determined by using spectrophotometer at wave length 260nm followed by another reading at the wave length 280 nm. 3 The ratio between the two readings was calculated, considering the dilution factor (10). 4 DNA concentration based on OD could be calculated as follow
DNA Conc. g/ml (or ng/ l) = A 260 in OD units x (50 g/ml/1 OD unit) x dilution factor
Calculating the ratio between OD 260 and OD 280 indicated the purity of DNA isolated which ranged from 1.8 2.0
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Subjects and Methods
5- Mutation Detection for KLF6 gene
Mutation detection of KLF6 gene (Exons1, 2, 3 and 4) were done using non isotopic polymerase chain reaction low ionic strength Single strand conformation polymorphism silver staining method (PCR LIS SSCP silver staining) as described previously (Jiang et al., 2002 and Maruya et al., 1996).
Principle:
Single strand conformation polymorphism analysis (SSCP), described in Orita’s Watershed paper in 1989 , has proven to be a useful tool for the detection of single nucleotide polymorphism (SNPs). It has been successfully used for detecting unknown mutations and for screening known mutations (Winterpacht et al., 1995 and Wenz et al., 1999). Also LIS SSCP is referred to as the low ionic strength single stranded conformation polymorphism procedure, and is based on the diversity in the electrophoretic mobility of ssDNA formed by heat denaturation in low ionic strength solutions. This method has been used to detect DNA polymorphism, including point mutations at a variety of positions in DNA fragments (Abba et al., 2001). In this technique, an amplified product of simple size is denatured and prevented from reannealing to its complement by dilution and addition of low ionic strength agent as sucrose. This forced each strand to reanneal to itself in a characteristic stable conformer. When these conformers are electrophoresed under native (non denaturing) conditions, the mobility of each strand is affected by its conformational structure. This structure depends on the sequence of nucleotides within the strand (Figure: 11).
Sequence variation within the PCR product, such as a point mutation, produces a different conformer, resulting in a change in electrophoretic mobility pattern. By comparing the strand mobilities between samples, genomic variation can be identified.
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Subjects and Methods
Figure (11): A schematic representation of the SSCP technique.
56
Subjects and Methods
LIS SSCP silver staining methodology:
(1) PCR amplification: The PCR for KLF6 gene (exon 1 4) was done as described previously by (Chen et al., 2003) where primer sequence for each exon was obtained and synthesized from (Integrated DNA Technologies, Inc). Table (4) show, the primer sequences, size of fragments amplified and annealing temperature for each exon.
Table (4): Primer sequences and the size range for klf6 exones used.
Size Annealing Exon range Primer sequences temperatur no. (bp) o C F: 5 ATgAAACTTTCACCTgCgCTCC 3 Exon 300 R: 5 TCTgAACCCCAAACAgCCgA 3 57 1
F: 5 TTTggTCgTCATggCAATCAC 3 Exon 290 57 2a R: 5 TTCCCgAgCTgACCAAAACTTC 3
F: 5 gTTTACCTCCGACCCCATTggC 3 Exon 320 59 2b R: 5 TCCAgggCTggTgCAATgCA 3
F: 5 CTgCgggTCAgTgAAgTCATggg 3
Exon3 275 56 R: 5 CATTCCCAAggCCCCACgCT 3
F: 5 AACACTggCAAgggATgggAACC 3
Exon4 300 R: 5 gCTATgCCgCTTCTTACAggACC 56 3
PCR conditions:
PCR was done in a total volume of 25 l for each sample containing primers for the amplified exon, four nucleotides, Taq DNA polymerase and its buffer which can be summarized as in Table (5).
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Subjects and Methods
Table (5): PCR conditions for KLF6 gene (exons 1 4) amplifications.
PCR mixture Volume/ Tube 10 X buffer contains tris HCl (pH 9.0), PCR enhancers, (NH 4)2SO 4, 20mM 2.5 l MgCl 2 dNTPs(dCTP,dGTP,dATP and dTTP) 0.5 l (10 mM) Forward primer (25 pmol) 0.4 l
Reversed primer (25 pmol) 0.4 l
Taq DNA polymerase (Exprime taq) 0.2 l (1 U) 5U/ l DNA 100ng (2.0 l)
dd H O 2 19.0 l
Total volume 25.0 l
First denaturation step, 5 minute at 95ºC followed by 35 cycles consists of denaturation for 30 seconds at 94ºC, annealing for 1 minute according to the temperature listed in table (4), and the extension for 45 seconds at 72ºC. Final extension for 10minutes at 72ºC.
(2) Agarose electrophoresis for PCR products :
PCR products were electrophoresed using agarose gel electrophoresis to check successfulness of the amplification process by PCR.
A Apparatus used:
1 Electrophoresis unit 8x11 cm (Bio RAD sub TM DNA cell). 2 Power supply (Hoefer Scientific Instruments). 3 Biodocument Analyzer (BioMetra, Germany). 58
Subjects and Methods
B Reagents:
1 Tris acetate EDTA buffer (TEA) 1X (As previously described). 2 Gel loading buffer (As previously described). 3 2% Agarose gel: Agarose …………………………………………………….2g, TAE (1X)……………………………………………… … 100 ml Ethedium bromide …………………………………… 0.5 g/ml, Agarose (2g) was added to 100ml TAE, cooked in a microwave for 4 minutes, and left to cool to 60 o C. Three l of ethidium bromide were added. The gel was casted in the tray of a submarine and after drying it was immersed in the electrophoresis unit.
C Procedure:
1 5 l of PCR product were added to 2 l loading buffer. 2 5 l of PCR marker (DNA ladder, Promega) were added to 2 l loading buffer. 3 The samples and PCR marker were loaded on 2% agarose gel in running buffer TAE (1X) and electrophoresed at 100 volt for 25 minutes. 4 PCR product was visualized under UV transilluminator using BioDocument Analyzer (Biometra, Germany) to indicate the successfulness of PCR product. The photographs were taken using the same analyzer to have permanent records.
(3) SSCP Analysis:
(A) Reagents : 1 40% polyacrylamide solution (stock solution) Acrylamide (DNA sequencing grade, Sigma)………...………….39g N, N methylene bis acrylamide………………………………….1g o Distilled Water (dd H2O) up to 100ml, the solution was heated at 37 C to dissolve the chemicals and stored in a dark bottle.
2 10X TBE buffer (stock solution) Tris base……………………………………………………….108g Boric acid……………………………………………………….55g EDTA 0.5 M, pH 8.0………………………………………….20ml The volume was completed to 1 liter by distilled deionized water (ddH2O). The buffer was stored at room temperature. 59
Subjects and Methods
3 12% polyacrylamide (Working solution) for non denaturing gel. Polyacrylamide 40% (stock solution)………………………....12ml TBE buffer (10X)……………………………………………....4ml The above solutions were mixed and the volume was adjusted to 40 ml.
4 1X TBE buffer (working solution) A volume of 100 ml of stock 10X TBE buffer was added to a volumetric cylinder and the volume was completed to 1 liter with ddH2O.
5 Ammonium persulfate (APS 10%) Ammonium persulfate…………………………………..………...1g The volume was adjusted to 10 ml using deionized distilled water. Aliquot the solution (1 ml/tube). Solution was stored at 4oC.
6 Lis SSCP loading Sucrose ………………………………………………………… 10g Bromophenol blue…………………………….……………..0.025g Xylene Cyanol………………………………………...... …..0.025g The volume was adjusted to 100ml using deionized distilled water. Aliquot the solution (1ml/tube). Solution was stored at 4 oC.
(B) Apparatus: * Electrophoresis cell (20x20cm) attached with cooling unit (Fisher scien, BioRAD) * Electrophoresis power supply (Biometra, Germany). High voltage power pack P30.
(C) Procedure: 1 Glass plates were cleaned and freed of soap residue, where 2 3 ml of 70% ethanol were applied to both plates and wiped dry to ensure that all soap had been removed.
2 The two glass plates were assembled with 0.8 mm side spacers, the sides of the plates were clamped to form a seal and put in casting unit.
3 For 20cm X 20cm gel the following solution was prepared, 12% polyacrylamide working solution…..……………….…40 ml APS (10%)…………………..……………………………200 l 60
Subjects and Methods
TEMED ………………….……………………………..…..15 l The solution was mixed gently and transferred to a syringe and the gel was poured along one side of the plates slowly to avoid air bubble formation.
4 The comb was inserted immediately.
5 The gel was allowed to polymerize. According to this procedure the gel could be stored for up to 24 hours at 4 oC. For prevention of dehydration during storage, 1X TBE buffer was added to the top of the gel, then, the gel was covered by saran wrap.
(D) Loading and running:
1 The gel was attached to the electrophoresis apparatus and the reservoir was filled with TBE buffer (1X). 2 The PCR product was diluted 1:2 with Lis SSCP loading buffer and denatured at 95 oC for 10 minutes. 3 The gel was pre run at 10W for 10 minutes.
4 20 l of denatured sample was loaded and the electrophoresis was carried out at 20W for 4 5 hours.
(E) Silver staining of the gel (according to Jiang et al., 2002 ) ● Reagents : All reagents were used in this part were of HPLC degree or analar grade. 1 Ethanol (10%) 10 ml of absolute ethanol (HPLC degree) was completed to 100 ml with dd H 2O. 2 Nitric acid (1%) 1 ml of conc. Nitric acid was added to dd H 2O to complete the volume to 100 ml. 3 Silver nitrate staining solution (0.25%) 0.25 g of silver nitrate (Analar grade) were dissolved to100 ml dd H2O and then 50 l of formaldehyde were added to the solution and kept in a dark bottle. 4 Developer solution Sodium carbonate ……………………………………………….3g Sodium thiosulfate (10%)...……………………………………20 l Formaldehyde ……………………………………………….200 l The volume was adjusted to 200 ml using ddH 2O.
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Subjects and Methods
5 Stopping solution Glacial acetic acid (Analar)…………………………….…..10ml The volume was adjusted to 100 ml using ddH 2O
●Procedure:
1 At the end of electrophoresis run, the power was turned off and the apparatus was disconnected from the power pack. The electrophoresis buffer was disposed and the gel mold was removed from the apparatus.
2 The gel was laid flat on the bench with uppermost small plate and the plates of the mold were slowly pried apart.
3 The gel was transferred to a Pyrex glass dish at which staining procedure performed.
Staining procedure was summarized as follow :
a Fixation: with 10% ethanol for 10minutes. b Sensitization with 1% nitric acid for 10 minutes. c Washing with ddH2O 2 times each for 2 minutes. d Silver Nitrate staining (0.25%) for 10 minutes e Washing with ddH2O for 30 second. f Developing with developing solution which added to the gel for 10 minutes, until the bands of interest were developed or determined. g Stopping the reaction with 10% glacial acetic acid for 5 minutes.
4 A piece of Whatman 3mm paper; slightly larger than the gel by 2 3 cm in both length and width; was centered over the gel.
5 A gentle pressure was applied, so that the gel becomes firmly attached to the rough surface of the paper covered with saran wrap and left to dry for 30 40 minutes, under vacuum on a gel dryer sited at 80 oC (Slab Gel Dryer, SGD 2000, Savant, Germany).
6 The gel was removed and saran wrap was peeled off.
7 The gel was scanned on computer scanner to save as photo in computer system.
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6-Sequencing of KLF6 gene (4-exons)
Principle:
DNA sequencing is the process of determining the exact order of the bases A, T, C and G in a piece of DNA (gene). In essence, the DNA is used as a template to generate a set of fragments that differ in length from each other by a single base. The fragments are then separated by size and the bases at the end are identified, recreating the original sequence of the DNA.
Automated DNA sequencing utilizes a fluorescent dye to label the nucleotides instead of a radioactive isotope. The fluorescent dye is not an environmentally hazardous chemical and has no special handling or disposal requirements. Instead of using X ray film to read the sequence, a laser is used to stimulate the fluorescent dye. The fluorescent emissions are collected on a charge coupled device that is able to determine the wavelength. In the present study, the cases that showed mutation of KLF6 gene using SSCP were reamplified and the PCR were purified using purification kit (Axygene Biosciences, USA) and sequenced forward strand only by the same primer used in SSCP.
(A) Purification of PCR product
The PCR products for KLF6 gene exons were purified or cleaned up using Axyprep PCR cleanup kits (Axygene Biosciences, USA).
●Principle :
The Axyprep PCR cleanup kits employ a special binding solution in combination with an Axyprep PCR column having an optimized chromatographic membrane to achieve high selectivity and recovery of linear DNA fragments. This product is designed to purity DNA fragments longer than 75 bp from PCRs and other enzymatic reactions, with an expected recovery of 70 90%. Each column has a binding capacity of up to 8 g. The purified DNA fragments are suitable for a variety of applications such as sequencing.
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Subjects and Methods
●Apparatus
* Microcentrifuge capable of 12.000 xg. * Heated water bath. * 95 100% ethanol.
●Procedure
A Preparation before experiment
1 95 100% ethanol was added to the concentrate Buffer W2 and was Mixed well and stored at room temperature. ..
2 Buffer PCR A was Checked to avoid precipitation before using. If precipitation occurs, it must be warmed at 65ºC to dissolve the precipitate. 3 Eluent was Pre warmed to 65ºC to improve elution efficiency.
B Protocol: (PCR Clean up Spin Protocol)
For each PCR product sample
1 100 l of Buffer PCR A was added to 20 l PCR products.
2 A PCR column was placed into a 2 ml microfuge tube. Pipetting the reaction from step 1 into the PCR column and the .centrifugation at 12.000Xg for 1 minute. .
3 The filtrate was discarded from the 2ml microfuge tube. Then, the PCR column was returned to the same tube. 700 l of Buffer W2 were pipette into the column and centrifuged at 12.000Xg for 1 minute.
4 The filtrate was discarded, and the PCR column was returned to the 2 ml microfuge tube. 400 l of Buffer W2 were added in the column and centrifuged at 12.000 Xg for 1 minute. . (These two washes with Buffer W2 were used to eliminate of salts)
5 The PCR column was transferred into a clean 1.5 ml microfuge tube. To elute the DNA, 30 l of eluent (pre warmed at 65 ºC) were added to the center of the membrane. After standing for 1minute at
64
Subjects and Methods room temperature, centrifugation at 12.000Xg for 1 minute was performed.
(B) Automated sequencing
After PCR clean up, the elute obtained undergoes automated sequencing by using BigDye Terminator v3.1Cycle Sequencing kit using Biosystem automated sequencer (The ABI PRISM 3100 Genetic Analyzer).
Automated DNA sequencing utilizes a fluorescent dye to label the nucleotides instead of a radioactive isotope. The fluorescent emissions are collected on a charge coupled device that is able to determine the wavelength.
The ABI PRISM 3100 Genetic Analyzer is a multi color fluorescence based DNA analysis system using the proven technology of capillary electrophoresis with 16 capillaries operating in parallel. The 3100 Genetic Analyzer is fully automated from sample loading to data analysis.
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Subjects and Methods
7- Loss of Heterozygosity (LOH)
Di , tri , and tetranucleotide repeats or "microsatellites" tend to be highly polymorphic and appear to be randomly distributed in the genome (Luty et al., 1990). The deleted chromosomal regions in tumors are pin pointed by examining a series of closely linked markers polymorphisms in the region and determining which of the markers heterozygous in the patients constitutional DNA have become homozygous in tumor DNA (i.e., which markers have lost on allele in the process tumorgenesis). This loss of heterozygosity in tumor DNA indicates that the normal tumor suppressor gene, as well as, polymorphic markers surrounding it, have been lost, leaving only the abnormal copy of the tumor suppressor gene (Jorde et al., 1999).
The loss of both copies of a tumor suppressor gene often involves a loss of heterozygosity in the chromosomal region where the gene lies in place of distinct maternal and paternal variants there is only one version of chromosome sequence or sometimes non at all. If a loss of heterozygosity is detected repeatedly at the same chromosomal site in many independently derived tumors, there is a strong presumption that this site normally harbors a tumor suppressor gene whose loss is instrumental in causing the tumors (Alberts et al., 1994).
LOH for KLF6 gene:
Loss of heterozygosity (LOH) on chromosome 10p15 regions (KLF6 locus) was detected by using three microsatellite markers which includes KLFM1, KLFM2, and KLFM4. The KLFM1, KLFM2, and KLFM4, flank the KLF6 locus by 40kb centeromerically, 10kb, and 20kb telomerically respectively.
The amplification of these three primers sequences were obtained as described previously (Reeves et al., 2004) and synthesized by (Integrated DNA Technologies, Inc.). The primer sequences and the size of the amplified fragments for the KLFM1, KLFM2 and KLFM4 are listed in Table (6).
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Subjects and Methods
Table (6): The primer sequences and the size of the amplified fragments for the KLFM1, KLFM2 and KLFM4 markers. Microsa Size Annealing tellite range Primer sequences temp. 0C markes. (bp) F:5 gAg ggAgTgAgg CTT TCT gTT 3 KLFM1 290 58 R:5 TTT CCA gCC CAC TgT CTT CTT gAC 3
F:5 ATg gCC CTg gTg ACT TCT TA 3 KLFM2 310 58 R:5 TAC TTg Cgg AgC gTg AgCC 3
F:5 gCATTAAgAATAgTgAAggC 3 285 KLFM4 53 295 R:5 gATgtgTTTggCTCAgggA 3
Reagents:
a Primers (25 pmol) b Taq DNA polymerase (Exprime Taq 5u/ l.) c 10X buffer (Exprime Taq) contains Tris HCl (pH 9.0), PCR enhancers, (NH4)2SO 4, 20mM MgCl 2 d 10 mM dNTPs mixture (dCTP, dGTP, dTTP and dATP)). e α [P 32 ] dCTP 10 Ci/ l (specific activity 3000 Ci/mmol, ICN, England).
PCR amplification for each marker
Amplification of KLFM1, KLM2, and KLM4 were done according to protocol shown in table (7).
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Subjects and Methods
Table (7): PCR conditions for KLF6 gene (LOH markers) amplifications.
PCR mixture Volume/ tube Final conc. 10 X buffer contain 20mM MgCl 2 1.25 l 1x Forward primer (25 pmol) 0.2 l 0.25 Reverse primer (25 pmol) 0.2 l 0.25 dNTPs (10 mM) 0.2 l 200 M α dNTPs P32 0.05 l 1 Ci Taq DNA polymerase (Exprime taq) 0.2 l 1 unit 5U/ul DNA 1.0 l 50 ng dd H 2O 9.4 l Total volume 12.5 l
The tubes were placed in a Master Cycler gradient (Eppendorf, Germany). The amplification program was as follows: 1 Denaturation at 95 oC for 5 minutes for 1 cycle. 2 35 cycles of denaturation for 30 seconds at 94 oC, annealing for 30 seconds at 58 oC for M1&M2 where it was 53 oC for M4, extension for 45 seconds at 72 oC. 3 Final extension for 10 minutes at 72 oC (1 cycle).
Running the gel
(A) Reagents:
1 40%polyacrylamide solution (38:2) (stock solution) Acrylamide (Acrylamide, SERVA, Heidelberg) …………..380g N, Nmethylenebisacrylamide……………….……………….20g Distilled water ………………………………………...600 ml The solution was heated at 37 oC to dissolve the chemicals. The volume was adjusted to 1 liter with distilled water and the solution was filtered through a nitrocellulose filter (Nalgen, 0.45 micron pore size) and stored in a dark bottle at 4 ºC.
2 10X TBE buffer (stock solution) Tris base………………………………………………….…108g Boric acid………………………………………………….…55g EDTA 0.5 M, pH 8.0………………………………………...20ml The volume was completed to 1 liter by distilled deionized water (dd H2O). The buffer was stored at room temperature. 68
Subjects and Methods
3 1X TBE buffer (working solution) A volume of 100 ml of stock 10X TBE buffer was added to a volumetric cylinder and the volume was completed to 1 liter with dd H2O.
4 6% polyacrylamide/ Urea (Working solution). Polyacrylamide 40% (stock solution)……………………..….75ml Urea …………………………………………………….….. 230g TBE buffer (10X)…………………………………….………25ml The volume was adjusted to 500 ml with deionized water and filtrated through a microcellulose filter (Nalgen, 0.45 micron pore size).The acrylamide/Urea solution may be stored for several weeks at 4oC.
5 Ammonium persulfate (APS 10%) Ammonium persulfate……………………………...…………..1g The volume was adjusted to 10 ml using deionized distilled water. Aliquot of the solution (1 ml/ tube) were stored at 4o C.
6 Gel loading buffer 98% deionized formamide……………………………………46ml EDTA 0.5M, pH8.0……………………………………………1ml 0.025% Xylene Cyanol FF……………………….……….0.0125g 0.025% Bromophenol blue………………………………..0.0125g
(B) Apparatus
* EC 160 DNA sequencing apparatus (Florida USA) OWL. * Two glass plates of slightly different sizes and one of them is notched. * Spacers made of thin flexible plastic (usually 0.4mm thickness). * High voltage power supply (Hoefer, Germany).
(C) Procedure:
1 Filling the glass plates * Glass plates and spacers needed for electrophoresis were washed in warm detergent solution, and rinsed thoroughly in tap water, followed by deionizing water. * The plates were held by their edges so that materials from the gloves or oils from hands were not deposited on the working surfaces of the plates.
* The plates were rinsed with ethanol and settled aside to dry. 69
Subjects and Methods
* The larger plate was laid flat on the bench and the two spacers were arranged in place along the sides then the smaller (notched) plate is placed in position, resting on the spacers. Three sided of the plates were clamped together with several large bulldog binder clips, of 5 cm length.
* The comb was placed into the open end of the gel mold and tested to see that it fits snugly.
* 250 l of 10% ammonium persulfate (APS) and 30 ul of N, N, N /, N / tetramethylene diamine (TEMED) were added to 50 ml of 6% polyacrylamide/urea (working solution).
* The solution was mixed gently and transferred to a syringe, the top of the plate was raised to angle 45 and slowly, the gel was poured along one side of the plate. The angle of the plate and the rate of addition were adjusted to avoid the air bubbles.
* After filling, the plate was laid on a flat surface and the sample will comb was inserted and clamped to prevent the gel forming between the glass plates and the sample comb. The gel was left overnight for polymerization at room temperature.
2 Loading and running buffer gel * After complete polymerization, the gel plate was mounted on an electrophoresis apparatus and sufficient 1X TBE buffer were prepared from stock solution (10X) TBE to fill the upper and lower chambers.
* 25 l of loading buffer were added to PCR product, mixed well and denatured by heating at 95 oC for 5 minute using Master cycler gradient (Eppendorfe, Germany), then chilled on ice immediately.
* The gel was pre run for 30 minutes at 55W before loading the samples.
* 6 l were loaded onto the gel in 1X running buffer.
* Electrophoresis was carried out at 55W for 2 3 hours at room temperature.
* After the electrophoresis, the gel was removed with Watmann 3M filter paper, covered with saran wrap and left to dry for 40minutes under vacuum on a gel dryer at 80 oC (Slab Gel Dryer, SGD 2000; Savant, Germany). 70
Subjects and Methods
* The gel was removed and the saran wrap was peeled off. * The dried gel was combined with x ray film enclosed in X ray cassette, then incubated overnight at 20 oC. * The x ray film was developed then the obtained image was analyzed.
3 Image analysis and interpretation of results
The samples were considered informative or heterozygous for each marker if two alleles could be identified with the normal tissue DNA. Loss of heterozygosity (LOH) was inferred from loss of one allele in the tumor DNA. The LOH was inferred when there was a visible reduction in the ratio of allele radiographic signal intensities in the tumor samples relative to that in the corresponding normal samples as visualized by two independent observers and by using BioDoc. Analyzer for scanning the films.
** Statistical analysis
The correlations between mutation at KLF6 gene and/or LOH in different markers used were analyzed in the three groups examined. Also mutation and/or LOH were analyzed with different clinicopathological parameters in each group using Chi square and Fisher's exact test. The association is considered significant when p≤ 0.05 (Bailey, 1994).
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Results
Results
In the present study, mutations at KLF6 gene (exons from 1 4) and LOH on chromosome 10p15 were detected in DNA isolated from 83 Egyptian patients. The patients were classified into three main groups as previously described in Subjects and Methods (see page 48). Groups included colorectal carcinomas CRC (38 patients), polyps group (23 patients) and inflammatory bowel disease (IBD) group (22 patients). The genomic DNA isolated from tissue samples of patients was separated on agarose gel to evaluate its integrity, purity and quantity. Figure (12) shows representative samples for the DNA isolated from the tissue biopsies of the CRC group.
Figure (12): Genomic DNA isolated from tissue biopsies for cases no. 1 8 of the CRC group. Separated on 1%agarose gel, stained with ethidium bromide and photographed under UV using Bio Doc Analyzer.
1 KLF6mutations
Mutations at KLF6 gene (exons 1 to 4) were detected using LIS SSCP silver staining technique followed by automated direct sequencing for cases that showed positive mutation using SSCP to confirm the results and to detect the exact pattern of mutations.
Our results revealed that about 39.5% of mutation (15 out of 38) was detected in CRC group whereas mutation recorded in polyps group was 26% (6 out of 23) and finally, it was 27.3% (6 out of 22) in IBD group. Statistically non significant variations was recorded between the mutations detected in the 3 main groups examined (p 0.3885). Figure (13) shows the percentage of distributions of mutation in the examined groups.
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40 35
30 25
20
15
10
% of KLF6 mutations KLF6 of % 5 0 C P IB RC oly D ps Examined groups
Figure (13): The percentage distribution for KLF6 mutation in the three different studied groups.
Correlation between KLF6 mutations and some clinicopathological factors: a Age:
The patients in the present study were classified into two main categories, the young age (≤ 50 years old) and old age (>50 years old). Non significant statistical variations between KLF6 mutations with the age in the 3 main groups were recorded where the mutation detected in CRC group was 38.5% (10 out of 26) and 41.7% (5 out of 12) in young and old ages respectively (p= .01). In polyps group, 28.6% (4/14) and 22.2% (2/9) of mutation were detected in both ages (p= 0.735) whereas the mutations detected in IBD group were 33.3% (5/15) and 11% (1/9) in young and old ages respectively (p= 0.2235). Figure (14) shows the percentage of KLF6 mutations in both ages of the groups examined.
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≤ 50y ≥ 50y
Figure (14): The percentage distribution for mutations in the two age categories in the three examined groups. b Gender:
The gender (no. of male and female) was estimated among the three different groups who shared in the present study. A correlation between mutations of KLF6 gene and the gender was calculated. Classifying the patients according to their gender showed a non significant correlation between them. In CRC group, about 16 of 38 (42.1 %) cases were males and 22 (57.9%) were females. Two only of 16 male cases (12.5%) exhibited KLF6 mutations where 13 of 22 females (59%) exhibited KLF6 mutations. This was statistically significant (p= 0.0037). In polyps group, about 18 of 23 (78.3 %) cases were males and 5 (21.7%) were females. Three of 18 male cases (16.6%) exhibited KLF6 mutations where 3 of 5 females (60%) exhibited KLF6 mutations. This was statistically significant (p= 0.05). In IBD group, about 17 of 22 (77.2 %) cases were males and 5 (22.7%) were females. Six of 17 male cases (35.3%) exhibited KLF6 mutations where there was no mutations detected in the 5 female cases in this group. Figure (15) is a histogram shows the distribution of KLF6 mutation in both sex for the examined groups.
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Results
Male 60 Female
50
40
m utations30 6
20
o f K L F
% 10
0 CRC Polyps IBD
Examined groups
Figure (15): The percentage distribution for mutations in the males and females in the three examined groups. c Site (location) of lesions:
According to the clinicopathological data sheet for each studied case, the colorectal lesions were subdivided into 2 main categories according to the site of lesions.
1 Right site includes (ascending colon, transverse colon, caecum and hepatic flexure). 2 Left site includes (descending colon, rectum, sigmoid and splenic flexure). Also there were some cases in groups II and III had lesions in the whole colon.
The statistical variation in the distribution of klf6 mutation results along the 3 studied groups according to the site of lesion was not significant. In CRC group, about 7 of 38 (18.4%) cases had lesions in the right colon and 30 (78.9%) had lesions in the left colon.
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Results
Three of 7 cases of right colon lesions (42.9%) exhibited KLF6 mutations where 12 of 30 cases of left colon lesions (40%) exhibited KLF6 mutations. This was statistically insignificant. In polyps group, about 6 of 23 (26%) cases had lesions in the right colon and 14 (60.9%) had lesions in the left colon. In addition, there were 3 cases had lesions in whole colon. Only one of 6 cases with right colon lesions (16.7%) exhibited KLF6 mutations where 5 of 14 left colon lesions (35.7%) exhibited KLF6 mutations. This was statistically insignificant. In addition there were no mutations detected in the whole colon lesion cases (3 cases). In IBD group, one of 22 (4.5%) cases had lesions in the right colon and 12 (55%) had lesions in the left colon. In addition, there were 4 cases had lesions in whole colon. There was no exhibited KLF6 mutation in the only case with right colon lesion where there were 5 of 14 left colon lesion cases (35.7%) exhibited KLF6 mutations. This was statistically insignificant. In the mean time, there were 3 of 4 cases with whole colon lesions (75%) exhibited KLF6 mutations in this group. Figure (16) is a histogram shows the distribution of KLF6 mutation in the different colon lesion sites for the three studied groups.
80 Right colon lesions Left colon lesions 70 Whole colon lesions 60
50
40
30
20 % of KLF6 mutations 10
0 Cancer Polyps IBD Examined groups
Figure (16): The percentage distribution for mutations of KLF6 gene in different lesion sites in the three examined groups.
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Results d Grade of cancer:
In cancer group, the degree of cancer invasion was pathologically determined. There were 3 cases with grade I colon cancer, 23 cases with grade II colon cancer, and 3 cases with grade III. Also, there were 9 cases their grade were not detected or unavailable. One of the 3 (33.3%) grade I cancer cases exhibited KLF6 mutation. Ten of 23 (43.5%) grade II cancer cases exhibited KLF6 mutation. Only one of 3 (33.3%) grade III cancer cases exhibited KLF6 mutation. In the mean time, 3 of 9 unknown cancer grade cases (33.3%) exhibited KLF6 mutation. This was statistically insignificant. Figure (17) shows the percentage of KLF6 mutations in different pathological grades of colon cancer cases in the cancer group.
Grade (I) 45% Grade(II) Grade(III) 40% Unknown grade
35%
30%
25%
20%
15% %of KLF6 mutations 10%
5%
0%
) ) I) (I (II e e (II d d e rade a a r r rad G G G nown g k Un Pathological colon cancer grade in the examined cancer cases
Figure (17): The percentage distribution for mutations of KLF6 gene in different pathological grades of colon cancer cases in cancer group. 77
Results e Dysplasia:
In IBD group, there were 19 of 22 cases (86.4%) pathologically were identified as ulcerative colitis cases. Ulcerative colitis (UC) subgroup was classified into 2 categories according to the presence or absence of dysplasia. Six of 19 ulcerative colitis cases (31.5%) exhibited dysplasia and 13 (68.5%) did not have dysplasia. Two of 6 (33.3%) dysplasia ulcerative colitis cases exhibited KLF6 mutations, where 4 of 13 (30.8%) ulcerative colitis cases with no dysplasia exhibited KLF6 mutations. This was statistically insignificant. Figure (18) shows the percentage of KLF6 mutations in the two categories of ulcerative colitis cases.
Dysplasia 33% Without dysplasia
33%
32%
mutations 32%
31%
% of KLF6 31%
30% Dysplasia Without dysplasia The examined ulcerative colitis categories
Figure (18): The percentage distribution for mutations of KLF6 gene in the two categories of ulcerative colitis cases. f Polyps size:
In polyps group, with consideration of polyps' size, there were 13 of 16 cases (81.3%) had polyps' size ≤ 1.0 cm, where 3 cases (19%) had polyps size ≥ 1.0 cm. About 5 of 13 polyps' size ≤ 1.0 cm cases (38.5%) exhibited KLF6 mutations where only one of 3 polyps size ≥ 1.0 cm cases (33.3%) exhibited KLF6 mutations. This
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Polyps≤ 1.0 cm 38% . Polyps≤ 1.0 cm
37%
36% 35%
34%
33% 32% %of KLF6 mutations 31%
30% Polyps≤ 1.0 cm Polyps≤ 1.0 cm Examined polyps group
Figure (19): The percentage distribution for mutations of KLF6 gene in the two size categories of polyps group.
2 Pattern of KLF6 mutation using sequencing analysis.
The bands or cases that showed abnormal pattern using SSCP technique were repeated twice and reamplified, purified and subjected for direct sequencing to confirm the results for SSCP and to detect the type and location of mutations. Our data indicated that:
(a) In CRC group:
There were 17 mutations observed in the 15cases that showed abnormal pattern in SSCP technique. Wheras two cases showed double mutations (case no. 1 and 38). Four of the 17 mutations detected were transitional type (23.5%) whereas 64.7% (11/17) were transversion type. One case only showed stop codon mutation (case 79
Results no. 10 at codon # 38) which leads to production of truncated or immature protein. Four mutations were silent (case no. 4, 19, 23 and 34) which did not lead to change of amino acid. One case was insertion of C at codon 223 (case no. 21) and other was deletion of A at codon 46 (case no. 30). Finally 10 mutations were missense type which leads to changes in the amino acids. The distribution of mutations between different exons was calculated. The highest frequencfy of mutation was recorded in exon 2 (14/17), 2 mutations at exon 3 and only one at exon 4. Figure (20 A) shows the percentage distribution of mutationin in different exons examined and figure (20 B) shows the percent of each type of mutations for KLF6 gene exons according to sequencing results in cancer group.
Exon1 Exon2 90 Exon3 Exon4
80 70
60 50
40
% of mutations of % 30
20 10
0 Exon1 Exon2 Exon3 Exon4 KLF6 exons
Fig (20 A): The percentage distribution for mutations of different exons of KLF6 gene in cancer group (GI).
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60 Missence
Non sence 50 Silent 40 Insertion
Deletion 30 Transition
Transversion 20
% ofdifferent types of mutations of types ofdifferent % 10
0 exon1 Exon2 Exon3 Exon4 KLF6 exons
Figure (20 B): The percent of mutations for KLF6 gene exons according to sequencing results in cancer group.
The pattern of mutations, codon number and amino acid changes in the examined colon cancer cases can be summerized in table (8). Figure (21) is the direct sequencing analysis for representative sample (case no. 1) of CRC for both normal and tumor tissues.
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Table (8): The pattern of mutations, codon number and amino acid changes in the examined colon cancer cases (GI).
Case no. KLF6 mutations
From To Type of Exon Codon a.a a.a mutation
2 S59R Serine Arg . Missense 1 2 I91L I Leu Leu Missense 4 4 R281R Arg . Arg . Silent 6 2 K66Q Lysine Gl n Missense 10 2 38 GLUT Stop Stop 11 2 S59R Serine Arg . Missense 16 2 S84R Serine Arg . Missense 19 2 S180S Serine Serine Silent 20 2 K73N Lysine Asparagin e Missense C 21 2 223 insertion 23 2 C36C Cyst Cyst Silent 27 3 R231I Arg . I Leu Missense A 30 2 46 deletion 34 2 C36C Cyst Cyst Silent 36 3 Y234C Tryp Cyst Missense 2 V199G Valine Glycine Missense 38 2 H200D HIS Aspartate Missense
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(A)
(B)
Figure (21): Detection of KLF6 gene mutations in CRC sample no. 1. (A) SSCP analysis for exon 2 for CRC samples (T1 T5) (B) Direct sequence analysis representative sample (case no. 1) of CRC for both normal and tumor tissues.
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(b) In polyps group:
There were 6 mutations observed in the 23 invistigated samples (26%) that showed abnormal pattern in SSCP technique. Two of the 6 mutations detected were transitional type (33.3%) whereas 66.7% (4/6) were transversion. One case only was silent mutations (case no. 2) which did not lead to change of amino acid. Finally 5 mutations were missense type which leads to changes in the amino acids. The distribution of mutations between different exons was calculated. The highest frequencfy of mutation was recorded in exon 2 (5/6) and only one at exon 4. Figure (22 A) shows the percentage distribution of mutationin in different exons examined and figure (22 B) shows the percent of each type of mutations for KLF6 gene exons according to sequencing results in polyps group.
90 % of mutations 80 70 60 50 mutations 40 30 20
% of% KLF6 10 0 Exon1 Exon2 Exon3 Exon4 KLF6 exons
Figure (22 A): The percentage distribution for mutations of different exons of KLF6 gene in Polyps group (GII).
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70 Missence 60 Silent
50 Transition Transversion 40
30 mutations 20
% of different types of 10
0 Exon 1 Exon2 Exon3 Exon4 KLF6 exons
Figure (22 B): The percentage of mutations for KLF6 gene exons according to sequencing results in polyps group.
The pattern of mutations, codon no. and amino acid changes in the examined colon polyp cases can be summerized in table (9). Figure (23) shows the SSCP silver staining analysis for exon 2 of KLF6 gene for polyps' tissues.
Table (9): The pattern of mutations, codon number and amino acid changes in the examined colon polyp cases (GII). KLF6 mutations Case From To Type of no. Exon Codon a.a a.a mutation 2 2 L79L Leu Leu Silent 6 2 S84R Serine Arg . Missense 7 4 S270C Serine Cyst Missense 9 2 I91L I Leu Leu Missense 14 2 S45R Serine Arg . Missense 15 2 G44R Glycine Arg . Missense
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Figure (23): SSCP silver staining analysis for exon 2 of KLF6 gene for polyps tissues. Normal pattern (two bands) was observed in cases 4, 5, 8 and 10, extraband in case # 6 indicate abnormal pattern as referred by arrow.
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(c) In IBD group:
There were 6 mutations observed in the 22 investigated samples (27.3%) that showed abnormal pattern in SSCP technique. Five of the 6 mutations detected were transversion type (83.3%) whereas the rest one mutant case was transitional type (16.7%). One case only was silent mutations (case no. 2) which did not lead to change of amino acid. Finally, 5 mutations were missense type which leads to changes in the amino acids. The distribution of mutations between different exons was calculated. The frequencfy of mutation distributions were recorded for each exon 2 and 4 (2/6) and one mutation at each exon 1 and exon 3. Figure (24 A) shows the percentage distribution of mutationin in different exons examined and figure (24 B) shows the percent of each type of mutations for KLF6 gene exons according to sequencing results in IBD group.
35 30 25 20 15 10
%of KLF6 mutations 5 0 Exon1 Exon2 Exon3 Exon4 KLF6 exons Exon1 Exon2 Exon3 Exon4
Figure (24 A): The percentage distribution for mutations of different exons of KLF6 gene in IBD group.
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35 Missence Silent 30 Transition 25 Transversion
20
15
10
5 % of different types of mutations 0 Exon 1 Exon2 Exon3 Exon4 KLF6 exons
Figure (24 B): The percentage of mutations for KLF6 gene exons according to sequencing results in IBD group.
The pattern of mutations, codon no. and amino acid changes in the examined colon IBD cases can be summerized in table (10). Figure (25) shows the SSCP silver staining analysis for exon 1 of KLF6 gene for IBD tissues.
Table (10): The pattern of mutations, codon number and amino acid changes in the examined colon IBD cases (GIII). KLF6 mutations Case From To Type of no. Exon Codon a.a a.a mutation 3 2 S112T Serine Tyro Missense 4 4 R281R Arg . Arg . Silent 8 4 S270C Serine cyst Missense 9 1 G11M GLUT Met Missense 11 2 F67I Phenyl I Leu Missense 15 3 R231I Arg . I Leu Missense
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Figure (25): SSCP silver staining analysis for exon 1 of KLF6 gene for IBD tissues. Normal pattern (two bands) was observed in Cases 5, 6, 9 and 12, extra band in case # 9 indicate abnormal pattern as referred by arrow.
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3 Loss of heterozygosity (LOH) at chromosome 10p15 region study
LOH analysis of the KLF6 gene in the present study was performed in 38 primary colorectal tumors, 23 polyps and 22 IBD using 3 microsatellite highly polymorphic markers (KLFM1, KLFM2 and KLFM4) which are located no more than 40 kb either telomeric or centromeric to the KLF6 gene as mentioned previously in Subjects and Methods part. Before doing the LOH analysis we optimized the PCR conditions for each marker without using the labeled radioactive phosphorus ( 32 P) to confirm the successful amplification of each fragment. Figure (26 a, b and c) shows the amplification for the 3 markers used and their exact size. The LOH data for each group studied can be summarized as follows: a Colorectal cancer group:
In cancerous group, the informative cases recorded for the 3 different markers used were high, where it was 86.8% for KLFM1 (33/38), 78.9% for KLFM2 (30/38) and 73.7% for KLFM4 (28/38). The overall LOH recorded in the 3 markers was about 28.9% (11/38) whereas LOH for individual marker was 15.2% (5 /33), 16.7% (5 /30) and 17.9% (5/28) for KLFM1, KLFM2 and KLFM4 respectively. Figure (27) shows the percentage of distribution of LOH for each marker and figure (28) shows representative cases for LOH analysis (cases # 23 and 27). In our study, only one case showed LOH in the 3 markers examined (case: 9), two cases had LOH at KLFM1/KLFM2 only (case: 6 and 38) as shown in figure (29). Figure (29) shows the individual distribution of LOH analysis in cancer group for the three microsatellite markers examined.
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(A): PCR products for KLFM1
(B): PCR products for KLFM2
(C): PCR products for KLFM4
Figure (26 A, B and C): PCR product for the 3 markers used in the present study (KLFM1, KLFM2 and KLFM4) without using radioactive 32 P in the reaction. M: is the ladder marker.
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KLFM1 KLFM2 KLFM4
19 18.5
18 17.5 17 16.5 LOH of % 16 15.5 15 14.5 KLFM1 KLFM2 KLFM4 LOH studied markers
Figure (27): percentage of LOH in cancerous group for the 3 different microsatellite markers used.
Figure (28): LOH study in cancerous group for KLFM1 marker. PCR product was separated on acrylamide after labeling with α dNTPs P32 , dried, exposed to x ray film. Heterozygosity was reported when 2 bands appear (indicated by arrows) in normal DNA (N). Loss of heterozygosity was reported when one band (allel) disappear (indicated by asterisk) in the tumor DNA. 92
Results
Figure (29): Individual distribution of LOH analysis in cancer group for the 3 microsatellite markers examined.
The variations in distribution of LOH analysis in some clinicopathological factors were examined in cancerous group; a non significant variations was reported between the LOH examined in either of age, gender, site of tumor and grade. About 34.6% (9/26) LOH was recorded in young ages (≤50 years old) where it was 25% (3/12) in old age (≥ 50 years old) and the p value was (p= 0.3918). About 18.8% (3/16) LOH was recorded in male where it was 36.4% (8/22) in female and the p value was (p= 0.5). About 28.6% (2/7) LOH was recorded in right colon lesion cases where it was 33.3% 93
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(10/30) in left colon lesion cases and the p value was (p= 0.975). About 21.7% (5/23) LOH was recorded in grade II cancer cases where it was not LOH recorded in either grade I cancer cases (0/3) and grade III cancer cases (0/3). In the mean time, about 11% (1/9) LOH was recorded in unclassified cancer cases, p value was (p=0.4547). Figure (30) shows the distribution of LOH for the 3 examined markers with different clinicopathological factors examined.
35% KLFM1 KLFM2 KLFM4 30%
25% gene 20%
15%
10% % of LOH for KLF6 for LOH of %
5%
0% Left Male Right GradeI Female gradeII
GradeIII
Unclassified cancer Unclassified Age(≥ 50 years old) old) years 50 Age(≥
old)Age years Age(≤50 Age categories Gender cancer grade Site of lesions Clinicopathological examined factors
Figure (30): The percentage distributions of LOH markers with different clinicopathological examined factors in cancer. b IBD group:
The incidence of LOH in IBD (includes ulcerative colitis, 19 cases and Crohn's disease, 3 cases) were recorded in the present study. The informative cases for the 3 markers examined were 81.8% (18/22), 86.4% (19/22) and 72.7% (16/22) for KLFM1, KLFM2 and KLFM4 markers respectively. Of the 22 cases examined, 8 had LOH (36.4%) in at least one of the 3 markers used. KLFM1 marker 94
Results showed 16.6% (3/18) LOH, KLFM2 showed 21% (4/19) and KLFM4 showed 12.5% (2/16). None of the Crohn's disease cases showed any LOH in the 3 markers used. The percentage of distribution of LOH for the markers examined is shown in figure (31), also figure (32) shows representative samples for LOH analysis. Case (7) showed LOH in KLFM1 and KLFM2 markers as in figure (33).
KLFM1 25 KLFM2 KLFM4
20
15
10 % of LOH of %
5
0 KLFM1 KLFM2 KLFM4 LOH studied markers
Figure (31): percentage of LOH in IBD group for the 3 different microsatellite markers used in IBD group
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KLFM4 KLFM2
8 7
*
* *
Figure (32):LOH study in IBD group for KLFM4 and KLFM2 markers. PCR product was separated on acrylamide after labeling with α dNTPs P32 , dried, exposed to x ray film. Heterozygosity was reported when 2 bands appear (indicated by arrows) in normal DNA (N). Loss of heterozygosity was reported when one band (allel) disappear (indicated by asterisk) in the tumor DNA.
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Figure (33): Individual distribution of LOH analysis in IBD group for the 3 microsatellite markers examined.
The variations in distributions of LOH analysis in some clinicopathological factors were examined in IBD group; a non significant variations were reported between the LOH examined in either of age, gender and site of lesions. About 37.5% (6/16) LOH was recorded in young ages (≤50 years) where it was 75% (3/4) in old age (≥ 50 years old) and the p value was p= 0.25. About 50% (8/16) LOH was recorded in male where it was 20% (1/5) in female and the p value was p= 0.25. The only case in right colon lesion had LOH (100%) where it was 66.6% (8/12) in left colon lesion cases and the p value was p= 0.5. Figure (34) shows the distribution of LOH for the 3 examined markers with different clinicopathological factors examined in IBD group.
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100% KLFM1 KLFM2 90% KLFM4
80%
70%
g e n e 60% 6
50%
40% of LOH for KLF
% 30%
20%
10%
0% Age(≤50 years Age(≥ 50 years Male Female Right Left old)Age old)
Age categories Gender Site of lesions
Clinocopathological examined factors
Figure(34): The percentage distributions of LOH markers with different clinicopathological examined factors in IBD.
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Results c Polyps group:
In the 23 polyps cases (16 adenomatous polyps, 3 FAB and 4 hyperplastic polyps), the informative cases for the 3 markers examined were about 86.9% (20/23), 78.3% (18/23) and 73.9% (17/23) for KLFM1, KLFM2 and KLFM4 respectively. The overall LOH detected was 52.2% (12/23) in at least one of the 3 markers used. The individual LOH showed 30% (6/20) in KLFM1; 33.3% (6/18) in KLFM2 and finally 17.6% (3/17) in KLFM4 markers. Two of four hyperplastic polyps examined had LOH in KLFM1 marker. No LOH detected in the 3 FAB cases. Figure (35) shows the distribution of LOH for the 3 markers used and figure (36) shows representative cases for LOH analysis. Case: 4 showed deletion in the 3 markers examined. One case only (case: 18) had deletion in both KLFM1 and KLFM2 markers. Figure (37) shows the individual LOH distribution for cases examined.
KLFM1 35% KLFM2 KLFM4 30%
25%
20%
15%
% of LOH 10%
5%
0% KLFM1 KLFM2 KLFM4 LOH studied markers
Figure (35): percentage of LOH in polyps group for the 3 different microsatellite markers used.
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KLFM4 MSI KLFM2
6 15
*
*
Figure (36): LOH study in polyps group for KLFM4 and KLFM2 markers. PCR product was separated on acrylamide after labeling with α dNTPs P32 , dried, exposed to x ray film. Heterozygosity was reported when 2 bands appear (indicated by arrows) in normal DNA (N). Loss of heterozygosity was reported when one band (allel) disappear (indicated by asterisk) in the tumor DNA.
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Figure (37): Individual distribution of LOH analysis in polyps group for the 3 microsatellite markers examined.
The variations in distributions of LOH analysis in some clinicopathological factors were examined in polyps group; a non significant variation was recorded between LOH for the 3 examined markers in different clinicopathological factors including age, gender, and site of lesions. About 50% (7/14) LOH was recorded in young ages (≤50 years) where it was 71.4% (5/7) in old age (≥ 50 years) and the p value was p= 0.5. About 62.5% (10/16) LOH was recorded in male where it was 80% (4/6) in female and the p value 0.9. All 6 right colon lesion cases had LOH (100% where it was 57.1% (8/14) in left colon lesion cases and the p value was 0.1. About 76.9% (10/13) LOH was recorded in polyps size ≤ 1cm cases, where there was no LOH recorded in the 3 cases with polyps' size ≥ 1cm and this correlation was significant at p value was 0.05. Figure (38) shows the distribution of LOH for the 3 examined markers with different clinicopathological factors in polyps group.
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50% KLFM1
KLFM2 45% KLFM4
40%
35%
30%
25%
20%
% of LOH for KLF6 gene 15%
10%
5%
0% Age(≤50 Age(≥ 50 Male Female Right Left polyp≤ 1cm polyps' size ≥ years old)Age years old) 1cm Age categories Gender Site of lesions Polps size
Clinicopathological examined factors
Figure(38): The percentage distributions of LOH markers with different clinicopathological examined factors in polyps group.
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Statistical relations between mutations and LOH in the KLF6 gene.
The distribution of klf6 status (mutations and LOH) in cancer group GI was summarized in figure (39). There were 21 cases had abnormalities in KLF6 gene (mutations and LOH) 55.3% and 17 cases had normal KLF6 gene (44.7%). Seventeen mutations were detected in 15 patients where 2 patients (case no. 1and 38) showed double mutations about (44.7%). LOH was detected in 11out of the 38 investigated samples (28.9%). There were 5 cases who showed klf6 mutation and LOH together and represented 13.2% of cases (case no. 6, 4, 27, 36 and 38). The clinicopathological data for these cases are shown in table (11).
KLF6 status in GI
LOH
29% Normal 44%
Mutations 44% Normal Mutations LOH
Figure (39): The distribution of KLF6 status results in GI
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Table (11): The clinicopathological data of some cases in GI.
No. Age Sex Grade Location Klf6 status mutation LOH 62 4 Female II rectum Ex.3 M2
Male 6 29 II sigmoid Ex.2 M1,M2
27 28 II rectum Ex.4 M4 Female
36 40 II rectum Ex3 M2 Female 38 rectum Ex.2 M1,M2
Non significant variations were observed between KLF6 mutations and LOH in this group while there were some statistical significances in this group between LOH markers and the mutation in exon 2. These data were summarized in table (12).
Table (12): statistical relations in GI
GI Relations p value
0.05٭٭ M1and exon2
Cancer Group 0.05٭٭ M4 and exon2
In group II (polyps): The distribution of klf6 gene status (mutations and LOH) in group II was summarized in figure (40). There were 13 cases had abnormalities in KLF6 gene (mutations and LOH) about 57% and 10 cases had normal KLF6 gene (43%). Six cases of mutations were detected in 23 patients (26%). LOH was detected in 12 out of 23 investigated samples about (52.2%). There were 5 cases who showed klf6 mutation and LOH together and represented 21.7% of cases (case no. 2, 6, 9, 14 and15). The clinicopathological data for these cases are shown in table (13).
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KLF6 status in GII
Normal LOH 43% 55%
Mutations 26% Normal Mutations LOH
Figure (40): The distribution of KLF6 status results in GII.
Table (13): The clinicopathological data of some cases in GII.
KLF6 status
Age Sex Location No. LOH Mutations
M2 2 56 Male Sigmoid Exon2
M4 6 53 Female rectum Exon2 (MSI)
Transverse 9 69 Male Exon2 M4
Transverse 14 60 Male Exon2 M1
Rectum 15 5 Male Exon2 M2
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There were significant statistical variations were observed between KLF6 mutations and LOH in this group (p value≤ 0.01).
Among the 22 samples included in GIII (IBD), the distribution of klf6 gene status (mutations and LOH) was summarized in figure (41). There were 11 samples had abnormalities in KLF6 gene (mutations and LOH) 50% and 11 samples had normal KLF6 gene (50%). Six mutations were detected in 22 patients (27.3%). LOH was detected in 8 out of 22 investigated samples (36.4%). There were 3 cases who showed klf6 mutation and LOH together which represent 13.6% of cases (case no. 4, 8 and 9).The clinicopathological data of these cases shown in table (14).
KLF6 status in GIII
LOH, Normal, 36% 50%
Mutation 27% Normal Mutations LOH
Figure (41): The distribution of KLF6 status results in GIII.
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Table (14): The clinicopathological data of some cases in GIII .
KLF6 status
Age Sex Location No. LOH Mutations
Recto M1,M4 4 45 Male Exon4 sigmoid Spelinic M2 8 30 Male Exon4 flexture 9 Recto 30 Male Exon1 M1 sigmoid
There were significant statistical variations between KLF6 mutations and LOH in this group (p value≤ 0.05).
A non significant statistical variation was observed between LOH and KF6 mutated samples as well as between Exon2 and KLM1 among the three studied groups (p value=0.1). Where a significant statistical variations between total LOH and exon2 mutated samples was observed at p value≤0.05 among the three studied groups.
There were significant statistical variations between gender and both KLF6 mutated samples (p value≤0.05) and LOH cases (p value≤ 0.01).
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------Discussion
Discussion
Colorectal cancer ( CRC) has been found to affect approximately one million people world wide (Reeves et al., 2004). (CRC) is also one of the most common human malignancies with more than 30,0000 cases both in the United States and in the European Union each year (lievre et al., 2005). It has been estimated that every American has a 6% lifetime risk of developing CRC. These statistics rendered CRC a major health concern in USA (Zhao et al., 2004). In contrast to these data, this disease seemed to be uncommon in developing countries (Magrath and Litvak, 1993), including Egypt where it is only the fifth most common cause of cancer death (Soliman et al., 1999). However, the percent of young onset colorectal cancer cases in Egyptian is strikingly high with more than one third of cases occurring under age of 40 years, and the age adjusted mortality rates in young Egyptians are likewise high. In addition, rectal cancer is frequently high (Soliman et al., 1997; 1999). Despite the different therapeutic approaches, the morbidity of colon cancer is still increasing (Rupnarain et al., 2004).
As in most solid tumors the malignant transformation of colonic epithelial cells is to arise through a multistep process during which they acquire genetic changes involving the activation of proto oncogenes and the loss of tumor suppressor genes (Vogelstein et al., 1988). Significant progress has been made in understanding the molecular mechanisms that lead to CRC (Kinzler and Vogelstein, 1996, 1998; Ilyas et al., 1999). Fearon and Vogelstein (1990) presented evidences for multisteps genetic model of colorectal carcinogenesis. This model was based on the understanding that CRC is the result of accumulation of genetic changes (mutations) in key genes, including the inactivation of tumor suppressor genes and aberrant activation of proto oncogenes. In support of the multistep genetic paradigm is the frequent presence of loss of heterozygosity (LOH) of specific chromosomal loci that harbor tumor suppressor genes (Goel et al., 2003).
Like in other malignant diseases, the investigation of the expression of tumor suppressor genes and oncogenes of colon cancer patients may reflect expression changes ongoing within the tumor, and thus open new approaches to the early diagnosis and thereby secondary preventions of colon cancer (Csontos et al., 2008).
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Selection of the target gene:
It is now generally accepted that colorectal cancer develops by genetic alterations. Analysis of the molecular mechanism makes it possible to develop a more targeted approach to prevention and treatment of this cancer (Takayama et al., 2006). There are two major pathways in colorectal cancer carcinogenesis. One is the chromosomal instability pathway (adenoma carcinoma sequence), which is characterized by allelic losses, and the other is the pathway involving microsatellite instability (MSI). However, other studies have suggested that colorectal carcinogenesis is not necessarily clearly divided into these two pathways. Other routs, including the serrated and epigenetic pathways, have been reported. There is also some cross talk among these pathways. Moreover, in the progression and metastasis steps of colorectal cancers, many more gene alterations are involved (Takayama et al., 2006). The relative contributions of the known or other potential tumor suppressor genes/oncogenes to the initiation and progression of malignant transformation is not fully characterized. In the mean time the mechanisms that underlie the deregulated cell growth in colorectal cancer remains the subject of intense investigation. In addition, mutation of the APC gene as an early initiating event is reportedly a less frequent finding in IBD related CRC (Harpaz & Talbot, 1996; and Wong&Harrison, 2001).
Kruppel like factor 6 (KLF6) is a ubiquitously expressed as zinc finger transcription factor that is part of a growing KLF family. The KLf family is broadly involved in cell differentiation and development, growth related signal transduction, and cell proliferation, apoptosis and angiogenesis (Bieker, 2001 and Black et al., 2001). The genetic alterations of the KLF6 gene have been identified in several human cancers, including hepatocellular carcinoma, prostate, gastric and colon cancers (Chen et al., 2003; Kremer-Tal et al., 2004; Cho et al., 2005; and Reeves et al., 2004). KLF6 has been identified as a tumor suppressor gene in several kinds of cancers because of its frequent genetic alterations, such as loss of heterozygosity and mutation, as well as its role in the functional suppression of cell proliferation (Cho et al., 2006).
On basis of functional analysis, wild type KLF6 up regulates the cell cycle inhibitor p21 in a TP53 independent manner and it suppresses growth, whereas tumor derived KLF6 mutants fail to up 109
------Discussion regulate p21 or suppress proliferation in prostatic and non small cell lung cancer cells (Narla et al., 2001; and Ito et al., 2004) . In addition, introduction of KLF6 disrupts the cyclin D1 cyclin dependent kinase 4 complexes and forces the redistribution of p21, which promotes G1 cell cycle arrest (Bebzeno et al., 2004). KLF6 also plays a role as an inhibitor of cell proliferation by counteracting the function of c Jun protooncoprotein (Salvin et al., 2004). These findings have led to hypothesize that genetic alteration of KLF6 gene might be one of the possible mechanism of KLF6 inactivation in colorectal cancers. In addition, Reeves et al (2004) reported that a high frequency (44%) of KLF6 somatic mutations had been founded in colorectal cancers. However, the relation between these genetic changes and different pathological stages from colorectal lesions to colorectal cancer is unclear until now.
Studying the early genetic changes that accompany the process of carcinogenesis is a major objective of a large number of research projects all over the world. Data from these studies are of great value in shedding light on the possible mechanisms that contribute to the changes of the preneoplastic cell to neoplastic cell. In the mean time, these early genetic changes can be used as markers for early prediction of the cancer onset. This guarantees early intervention that may save many lives. In this respect, there are some evidences that point to the existence of a genetic susceptibility to colon cancer and the disruption of functional activity of the KLF6 gene may be involved in the early stages of the disease. Relative of patients with both inflammatory bowel disease (IBD) and colorectal cancer had increased risk of developing colon cancer (Askling et al., 2001). In the meaning time; in non small lung cancer; there were no associations between KLF6 expression and clinicopathological parameters (Ito et al., 2004). Mutations of the KLF6 gene were reported in sporadic colon cancer as well as in IBD related colon cancer. In addition, in IBD related cancer cases in which histological evidence of dysplasia has been demonstrated, the pattern of KLF6 allelic loss in inflamed tissue was almost identical to that seen in the tumor (Miyaki et al., 2006).
The role of KLF6 gene inactivation in colorectal cancer is still unresolved (Lievre et al., 2005). So the combination of KLF6 mutations and KLF6 LOH in one study may contribute to a particular stage in multisteps colorectal carcinogenesis. Due to the limited studies on KLF6 gene in preneoplastic lesion of colorectal cancer, its
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------Discussion role in the progression of CRC is still unclear. In the present study, and in attempt to elucidate its role in the early event of neoplasia in colorectal cancer in an Egyptian populations, we examined mutations and loss of heterozygosity for pre neoplastic (IBD, polyps) and neoplastic lesions of colorectal patients.
A- KLF6 Mutation:
In the present study, low ionic strength single strand conformational polymorphism silver staining (LIS SSCP silver staining) technique followed by automated sequencing were applied for the detection of KLF6 gene mutations along its four exons of DNA isolated from tissue biopsies for patients with colorectal lesions and cancers. The mutations of KLF6 gene (exons 1 4) have been found in 27 of total 83 colorectal lesion including cancer samples (about 33%). In previous studies, the genetic alterations of the KLF6 gene were applied to cancer cases only and the results which have been observed were 55% of prostatic cancer (Narla et al., 2001), 44% of colorectal cancer (Reeves et al., 2004), 15% of hepatocellular carcinoma (Kremer-Tal et al., 2004) and 9%of astrocytic glioma (Jeng & Hsu, 2003). Also, there have been no available studies for pre neoplastic lesions of colorectal cancer (Cho et al., 2006).
1- KLF6 mutations in cancer patients (GI)
High frequency of mutations at KLF6 gene was detected in colorectal cancer samples most of the mutant cases were somatic missense mutations which alter the type of amino acid (10 cases), four mutations were silent, one was stop codon, one was insertion and the other was deletion in the present study (39.5%, 15/38). The high frequency of mutation in this study is in accordance with previous studies for colorectal carcinomas (Reeves et al., 2004 and Cho et al., 2006), who reported high frequency of mutations in KLF6 gene. Our present data also appear to be highly discordant with that reported by Lievre et al (2005) who failed to find any mutations in CRC patients. It was also reported such discrepancies in incidence of KLF6 mutations in prostate cancer (Narla et al., 2001 and Chen et al., 2003), hepatocellular carcinomas (Kremer-Tal et al., 2004 and Song et al., 2006) and astrocytic glioma (Koivisto et al., 2004). The reason for these discrepancies may be due to the difference between sample sets in different studies, racial differences and the mutational spectrum in addition to the different genetic and environmental 111
------Discussion background. The risk for false positive in the present study was excluded as the DNA used was isolated from fresh frozen tissues in addition to that the mutant samples was repeated 3 times using different conditions followed by sequencing analysis which increases the sensitivity of SSCP technique for detection of mutation.
On the other hand, Dong (2006) reported that germline SNP in KLF6 gene generates a functional SRp40 DNA binding site and increases transcription of three alternatively spliced KLF6 isoforms, which produce variant KLF6 proteins that are mislocalyzed to the cytoplasm and antagonize wild type KLF6 function. Finally, Cho and coworkers (2006) suggested that genetic alterations of KLF6 gene might be one of the possible mechanisms of KLF6 inactivation in colorectal cancer.
2- KLF6 mutations in colorectal polyps' patients (GII)
Pathologically, polyps are the early events (colon lesions) that may lead to colon cancers development. Polyps' lesions are considered as pre malignant events according to adenoma carcinoma sequences. For this reason, the present investigation was designed to answer the question whether mutations of klf6 gene (especially exon 2) in adenomatous polyps are early indicator for colorectal carcinogenesis.
The present data indicated that about 26% (6/23) had KLF6 mutations at different exons examined. All of the mutant cases were adenomatous polyps cases and none of the 3 FAB cases exhibited any KLF6 mutation. This finding may indicate its involvement in the mechanism of carcinogenesis of sporadic colorectal cancer rather than familial one. In the mean time, this finding does not preclude KLF6 gene dysregulation in the other types of polyp's subgroups (FAP and HP), in which the dysregulations of KLF6 may occur as a result of promoter hypermethylations as stated by Yamashita et al (2002) and/or dysregulated alternative splicing as stated by Narla et al (2005).
3- KLF6 mutations in IBD patients (GIII)
Sequencing the KLF6 gene revealed that, KLF6 gene mutations were restricted to the ulcerative colitis cases only. There were 6 cases out of 19 investigated ulcerative colitis cases (32%) had
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------Discussion
mutations at least in one of KLF6 gene exons. Five of 6 cases were missense mutations which lead to change in the amino acid and one was silent. None of the 3 Crohn s diseases cases showed any mutations
All cases that showed KLF6 mutations were suffering from whole colon ulcerations. Previous studies have revealed that, ulcerative colitis is a chronic inflammatory disease that produces reactive oxygen and nitrogen species and increases the risk of colorectal cancer (CRC) (Hussain et al., 2000). The colons of UC patients show uniform and continuous inflammation of the mucosa. The inflammatory reaction causes epithelial damage, including multiple regions of ulceration and hemorrhage. While adenomatous polyps are considered to be the major precursor of sporadic CRC (Fearon, 1994), ulcerative colitis–associated neoplasm involves the development of epithelial dysplasia that may affect large regions of mucosa (Hussain et al., 2000). Several studies have indicated that patients with ulcerative colitis have a 2 8.2 relative risk of colorectal cancer compared with the normal population, accounting for about 2% of colorectal cancers (Hardy et al., 2000).
The results of ulcerative colitis subgroup cleared that, all mutated cases were male and 33.3% of mutations were at age less than 50 years old. Eleven percent of mutated samples were at age more than 50 years old . Askling et al (2001) reported that inflammatory bowel diseases (IBD) and colorectal cancer might share a common cause and, therefore, relatives of patients with IBD could be at increased risk of this malignant disease . Also, Rhodes (1996) had suggested the existence of a genetically mediated glycosylation defect in both types of disease; therefore, he suggested that relatives of patients with IBD could be at increased risk of this malignant disease. According to Rhodes ’ hypothesis (1996), ulcerative colitis, Crohn’s disease and colorectal cancer are all caused by inherited defective glycosylation. This defect would, on the one hand, act to increase the binding of lectins, leading to increased cell proliferation and cancer, and on the other hand, result in a weak mucus that would be dependent on the presence of environmental factors such as smoking and bacteria, which lead to either Crohn’s disease or ulcerative colitis. In addition to that, Satsangi and his coworkers (1996) stated that studying results of patients with IBD provided an evidence for a linkage close to a genetic region encoding susceptibility to colorectal cancer . Beside this; ulcerative colitis is common in young people as found in Egypt perhaps
113
------Discussion due to changes in lifestyle (Hussain et al., 2000). On the other hand, in a register study, Fonager and his colleagues (1998) did not record an increased risk for colorectal cancer in relatives of patients with IBD while Askling et al. (2001) demonstrated an increased risk of colon cancer in relative of patients with colon cancer and IBD.
It should be noted that, however in the IBD group, KLF6 gene mutations were exclusively restricted to patients with ulcerative colitis. This may denote a negligible role of KLF6 gene mutations in malignant transformations of lesions associated with Crohn's disease. The inability of detection of KLF6 gene mutations in these patients may be due to the fewer number participating in the present investigation. KLF6 gene mutations in CRC associated with IBD patients have been studied by Reeves et al (2004). They reported that such mutations were reported in 60% of patients with IBD related tumor and in 35% of patients with sporadic colon cancer. This emphasizes the role of the KLF6 in IBD malignant transformation.
Most of the mutations at KLF6 gene in the present study was localized in exon 2 as in cancer group (14 out of 17), polyps (5 out of 6). These results reveal that exon 2 is the most affected exon in colorectal cancer and polyps cases so it may play a role as a marker for colon cancer diagnosis. Chen et al (2003) reported that according to the available human genome sequences in Gene Bank of chromosome 10, the KLF6 gene has four exons. The size for exon 1 to 4 is 218bp, 574bp, 124bp, and 525bp respectively. The 83% of mutations occurring in exon2, and clustering of mutations in exon2 were also reported in previous study for Narla et al (2001). The majority of exon 2 encodes for the trans activation domain of the KLF6 protein, which varies greatly among different KLF members (Philipsen and Zuske, 1999). Vax et al (2003) analyzed 60 pituitary adenomas with direct sequence analysis of exons 2 and 3 of the KLF6 gene because they considered that these are the regions where most of the previously described mutations are expected to be located.
About 60% of mutations in KLF6 gene were deleted type in IBD related CRC as reported previously (Reeves et al., 2004). This finding in addition to the previous data refers to the role of KLF6 gene mutations as an early event in UC CRC pathway.
Four of 17 mutations detected in cancer group affected serine residues, three cases were from polyps group and 2 cases were from
114
------Discussion ulcerative colitis group. It was reported previously that the serine residues in the transactivation domain of KLF6 gene may be important for its function and the mutations at serine residues affects the transcriptional activity of KLF6 gene.
In the present study, the distribution of KLF6 mutations were detected in 15 of 38 colorectal cancer patients group (39.5%); 6 of 23 colorectal polyps patients group (26%) and 6 of 22 colorectal IBD patients group (27%). However, there was no statistically significant difference in KLF6 mutations in the three studied groups. This supports the assumption that the KLF6 gene mutations may be an early event in the causes of premalignant to malignant transformations in colonic lesions.
B- Loss of heterozygosity in chromosome10p15 locus (KLF6 allelic loss):
Allelotype studies have shown that specific chromosome regions are frequently deleted in many tumor types and the frequent loss of heterozygosity (LOH) at certain chromosomal regions are a hallmark of the existence of a tumor suppressor gene. Therefore, identifying consistent areas of chromosomal deletion in a particular tumor’s DNA may indicate regions that harbor tumor suppressor gene at this locus, which contributes to carcinogenesis in that tissue (Cho et al., 2006). In a previous allelotype analysis on all the chromosomes of colorectal cancer, frequent LOH has been reported on the short arm of chromosome 10 (Shima et al., 2005). Because the KLF6 gene is located on chromosome 10p15 (Onyango et al., 1998) and is one of the possible tumor suppressor genes in human cancers (Cho et al., 2006), the present study was concerned with determinations of loss of heterozygosity of KLF6 gene.
In the present study, from a total of 83 DNA samples, 80 DNA samples were examined for LOH at short arm of chromosome 10 at locus 15(10p15). This was done by using α dCTPs P 32 LOH PCR for 3 different LOH markers KLFM1, KLFM2 and KLFM4 which flank the KLF6 locus by 40kb centeromerically, 10kb, and 20kb telomerically respectively (Reeves et al., 2004). The remaining 3 samples were excluded from this experiment because their corresponding normal biopsies were not available. From a total of 80 DNA samples investigated, 31 samples show allelic loss at least in
115
------Discussion one of the 3 markers used (39%) and these cases distributed among 3 studied groups with the following percentage 29, 52 and 36 respectively. The variation in distribution of LOH results (+,─) along the 3 studied groups was statistically insignificant at p value=0.1, in the mean time the variation in distribution of results of LOH markers individually (M1, M2and M4) along the studied groups was also not significant. Twenty out of the 71 investigated samples (28%) show allelic loss in KLFM1 markers, and 15 out of 67 investigated samples (22%) show allelic loss in KLFM2 markers where in KLFM4, 10 out of 63 investigated samples (16%) showed allelic loss. There was one case showed allelic loss in the three markers (from cancer group) and another case in polyps group also, and there were some cases who showed allelic loss in 2 of the three investigated markers (5 cases with percentage 6%). The over all allelic losses for the three markers examined in the present study showed high frequency in the 3 groups where they were 29%, 36% and 52% in cancer, IBD and polyps groups respectively. Previous studies have detected high percentage of LOH at KLF6 gene of invasive colorectal cancer ( Reeves et al., 2004; Cho et al., 2006 ), gastric cancer (Cho et al., 2005) and gliomblastoma (Camacho-Vanegas et al., 2007). In the present study, LOH was detected in preneoplastic lesions including adenomatous polyps and IBD in addition to neoplastic lesions. Previous study has reported allelic loss of KLF6 in inflamed colonic epithelium which was similar to that seen in adjacent tumors. Taken together, these finding suggested that KLF6 loss may be an early events in the pathogenesis of colorectal cancer through either adenoma carcinoma sequence or UC CRC pathways. No LOH was detected in the 3 FAB cases examined in the current study which indicates that KLF6 loss is specific to the sporadic colorectal carcinogenesis and the mechanism is different from those in FAB as reported previously (Miyaki et al., 2007).
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------Discussion
C- KLF6 mutations and LOH
In the light of combined results of mutations and LOH for KLF6 gene in this study, gave a possible strong evidence for the role of KLF6 in the development and progression of colorectal lesion to cancer. This was consistent with Cho and his coworkers (2006) who suggested that genetic alterations of KLF6 gene might be one of the possible mechanisms of KLF6 inactivation in colorectal cancer.
It was observed that 4 of the 11 that have LOH in cancer cases were associated with mutation in KLF6 exons, 4 out of 8 deleted cases in IBD group showed mutations and finally, 3 out of 12 deleted polyps groups had mutations. Absence of mutation in one allele and loss of the second allele was previously reported in different studies which is quit different from classical tumor suppressor genes that are inactivated through an LOH in one allele and mutation in the other. There is a possibility that single allele of KLF6 may lead to tumor progression through a haploinsufficiency mechanism as observed in glioplastoma (Narla et al., 2001) or may be there is another tumor suppressor gene close to KLF6 and is involved rather than KLF6 itself.
Interestingly, case 9 from tumor and case 4 from adenomatous polyps showed deletion in the three markers examined (M1, M2 and M4) which mean that both cases may undergo whole chromosomal deletion. Also, case 6 and 38 from tumor samples and case 7 from IBD and case 18 from adenomatous polyps showed deletion in both M1 and M2 markers which suggest that there is a common deletion in this region is involved in carcinoma as well as in both IBD and sporadic adenomatous polyps. No clear significant association was observed between genetic alterations (LOH/mutations) in KLF6 of this study with different clinopathological parameters such as gender, age, site of tumor, grade and polyps size and further studies are needed to clarify it. See figures (29, 33 and 37 respectively).
In the mean time, two cancer cases (1 and 11) have mutation at the same codon (S59R) at the same time both are females, early age (13 and 38 years), the tumor in the rectum in addition a deletion at M4 marker was detected suggesting biallelic inactivation through missense mutation in one allele and loss of the remaining allele and also may indicate that this codon is hotspot of KLF6 gene mutation for such type of cancer. Case number 1 and 9 from cancer and polyps
117
------Discussion group respectively showed the same mutation (I91L), case number 27 and 15 from cancer and ulcerative colitis groups shared the same mutation (R231I) which may refers that they have the same etiological and environmental factors. See tables (8, 9 and 10).
Conclusion :
The data from the present work demonstrated the possible contribution of the KLF6 gene (mutations and LOH) in the development of colorectal cancer among Egyptians. In this respect, these data shed some light on the following issues:
1 Mutations of KLF6 may be involved in the development of a subset of colorectal cancer. Detecting mutational sites differing from that detected in western populations may be characteristic of Egyptian CRC due to environmental and genetic factors.
2 One of the important findings reported in the present study was the extremely high incidence of exon 2 mutations in all colorectal lesions.
3 Most of the mutations reported are either of transversion and/or missence types.
4 The selected LOH markers in the present study can be considered as early detection markers for colorectal cancer cases as well as in pre malignant lesions cases.
5 KLF6 gene alteration is involved in the progression of Egyptian colorectal carcinogenesis from both sporadic adenomatous polyps and ulcerative colitis pathways.
Recommendation:
In view of the previous findings, it is recommended that:
1 Cases who have pre neoplastic colon lesions in which the KLF6 gene has mutated or lost its heterozygosity should experience more frequent colonoscopic examinations for the detection of doubtful malignant changes.
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2 Since, KLF6 may be involved in the pathogenesis of colorectal lesions and cancer, so it may be an attractive candidate for gene therapy approaches.
3 Further studies are needed to elucidate the possible role and mechanism of KLF6 gene mutations reported here and inactivation of its protein in different colorectal lesions.
119
------Summary
Summary
Colorectal cancer remains a significant contributor to the world’s health burden. Early diagnosis and treatment can guarantee long survival of the patients. Early diagnosis is hampered by fact that most of the symptoms of early colon cancer resemble those associated with other diseases such as diarrhea and hemorrhages.
Considerable progress has been made through different studies focusing on investigating the changes of colon cancer at the molecular level. These studies shed some light on the identification and characterization of the genetic changes involved in colorectal cancer transformation. Data from these studies supported the concept of multistage carcinogenesis as being a consequence of multiple genetic alterations that are accumulated in cancer cells . Despite the enormous data reported in this respect, the molecular mechanisms underlying the dysregulated cell growth in colorectal cancer still remain the subject of intense investigation .
Like in other malignant diseases, the investigation of the expression of tumor suppressor genes and oncogenes of colon cancer patients may reflect expression changes ongoing within the tumor, and thus open new approaches to the early diagnosis and thereby secondary preventions of colon cancer .
Kruppel-like factor 6 (KLF6) is a ubiquitously expressed as zinc finger transcription factor that is part of a growing KLF family . The genetic alterations of the KLF6 gene have been identified in several human cancers, including hepatocellular carcinoma and prostate, gastric and colon cancers . KLF6 also has been identified as a tumor suppressor gene in several kinds of cancers because of its frequent genetic alterations, such as loss of heterozygosity and mutation, as well as its role in the functional suppression of cell proliferation. There are circumstantial evidences suggest that the disruption of functional activity of KLF6 gene associates the early events in colon carcinogenesis. The combination of KLF6 mutations and KLF6 LOH in one study may contribute to the understanding of the stages in multisteps colorectal carcinogenesis and provide a new marker for the early detection of colorectal cancer.
The main objective of the present study was to detect mutational changes of KLF6 tumor suppressor gene and to study the
- 120 ------Summary
loss of heterozygosity (LOH) markers at chromosome 10p15 (KLF6 locus) from colorectal lesions known to predispose to colorectal cancer in Egyptian patients.
The patients included in this study were 83 presented with different indications for colonoscopic examination. Selecting patients with colorectal cancer or colorectal pre-cancerous lesions was done according to the results of tissue biopsy from lesion and adjacent normal.
The patients were classified into three main groups; I- Cancerous group Thirty eight of patients were diagnosed as adenocarcinoma. II -polyps group : This group contained 23 patients which were diagnosed as Adenomatous polyps (16 cases), Familial Adenomatous polyps (FAP) (3 cases) and Hyperplastic polyps (4 cases). III- Inflammatory Bowel Diseases (IBD) Include 22 patients which were diagnosed as ulcerative colitis (19 cases) and Crohn's disease (CD) (3 cases).
The DNAs were extracted from the tissue samples. Genomic DNA isolated from tissue samples was electrophoresed using agarose gel electrophoresis to check successfulness of isolation and its purity. The purified DNAs were subjected to the following estimations:
1- Detection of KLF6 mutations Mutation detection of KLF6 gene (Exons1, 2, 3 and 4) were done using non-isotopic polymerase chain reaction-low ionic strength- Single-strand conformation polymorphism- silver staining method (PCR-LIS-SSCP-silver staining). The mutated samples were subjected to direct sequencing to detect the type of mutations and codon location at which mutations were occurred.
2- Determination of LOH in the separated DNA samples. Loss of heterozygosity (LOH) on chromosome 10p15 regions (KLF6-locus) was done using three microsatellite markers including KLFM1, KLFM2, and KLFM4.
- 121 ------Summary
Data from the present study could be summarized as follows:
In cancerous group : There were 21 cases had abnormalities in KLF6 gene (mutations and LOH) 55.3% and 17 cases had normal KLF6 gene (44.7%). Seventeen mutations were detected in 15 patients where 2 patients showed double mutations (44.7%). LOH was detected in 11 out of the 38 investigated samples (28.9 %). There were 5 cases that showed klf6 mutation and LOH together and represented 13.2% of cases.
In polyps group : There were 13 cases who had abnormalities in KLF6 gene (mutations and LOH) 56.5% and 10 cases had normal KLF6 gene (43.4%). Six cases of mutations were detected in 23 patients (26%). LOH was detected in 12 out of 23 investigated samples (52.2 %). There were 5 cases that showed klf6 mutation and LOH together and represented 21.7% of cases. In inflammatory Bowel Diseases (IBD) There were 11 samples who had abnormalities in KLF6 gene (mutations and LOH) 50% and 11 samples had normal KLF6 gene (50%). Six mutations were detected in 22 patients (27.3%). LOH was detected in 8 out of 22 investigated samples (36.4%). There were 3 cases who showed klf6 mutation and LOH together which represent 13.6% of cases.
In conclusion, our data highlight; for the first time; the role of KLF6 gene in the progression of Egyptian colorectal carcinogenesis where the results suggest that KLF6 gene alteration is involved in the progression of Egyptian colorectal carcinogenesis from both sporadic adenomatous polyps and ulcerative colitis pathways. Detecting mutational sites differing from that detected in western populations may be a characteristic of Egyptian CRC due to environmental and genetic factors.
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- 145 - Arabic summary
ن ا ن وا ا اع ا ن ا و ا ب ا ة ن ا ا ، آ ا ١١ ٢ % ت ا ا دول ق ا آ ر . و ض ن ا ن وا ازد د د ا ت ا آ ازد د د ا ت ذوى ا ت ا ا ة ( ا ٤٠ ) د ا ت . و ه ا ت ا ة ، و ى ذ ا ا ا ة و ىا رة ا ا او ا ا ى ا ا ات . آ ان ا ض ن ا ن وا ا ا ا ل وا ا ات ا اض ا ن ا . و ا ان ن ا ن وا ا آ ا دة ا ا ان ا آ ف ا ج ور ا ان ا ٥٠ % ا ا ا ا . . و ن ا آ ف ا ا ا ق ا ة ن ا ن وا . .
ض ن ا ن وا ن ا ل ا ا ا ة ات و ه ة ا ت ا ا ا ات ا ى ا ا ت ا ة ورام oncogenes و ت ا ورام . و KLF6 ا ت ا ورام ا ا ورام ا ، وه ك ا ا وا را ت ا ه ا ا ا ان ا ط KLF6 او ا و ا ر ا ا ب ا و او ا ة ا ل ا ض ن ا ن وا . .
ا ا را ا ى ا KLF6 آ ا ت ا ورام وآ س م ا ا ام د ا ا ا و م ر ١٠ . و دور ه ا ا ا ل ا ا ا ا ض ا ت ا ن و ا ض وآ ا ض ن ا ن وا . . و ل ذ دور ه ا ا ا ل ا ا ا ا اض ا ن وا . ه ا را ء وع ASTR ا ل اآ د ا ا ا ر ( ١٠٥ ). ).
ا ا را ٨٣ ا ن وا ا ا ا ام ر ا ن وا وذ ة ا ز ا ا آ ا ا ه ة ا ة ٢٠٠٥ ٢٠٠٧ . و ا ل ت ا ا ن وا ا وا آ
١ ١
Arabic summary
ه ء ا ا ا . آ ت ا و ا ا ت آ : : ا ــ ا و و ا ٣٨ ن ا ن وا . .
ا ـــ ا ـ و ا ٢٣ ا وا ا . .
ا ــ ا ـ و ا ٢٢ ا ت ا . .
• ا ص ا ا وى أ.ن.د( ) ا ه ا را . . • ا ا ات اآ ت ١ ٤ KLF6 س ا ف ا ا دى ).أ.ن.د( sequencing ل .د( ن أ. ) أ. ن ت ا ا ة و . . • س م ا KLF6 ام ا ا و د ا ر ا ٣٢ ا ا ة ا د آ ا ت. وذ ام د ا ا ا و م ر ١٠ وه KLFM1, KLFM2 & KLFM4 . .
و أو ا : : ا ا و : : ١٧ ة KLF6 ١٥ ن ا ن وا ، ان ه ك ن ن . و ات KLF6 ت ن ا ن وا ٤٤ ٧ %. م ا ٢٩ ٧ % ا ت ( ١١ / ٣٨ ). ).
ا ـــ ا ـ : و أ ٦ ات KLF6 ا ن وا ، و ات KLF6 ٢٦ %. م ا ٥٢ % ا ت ( /١٢ ٢٣ ). ).
ا ــ ا ـ : آ ٦ ات KLF6 ا ت ا ن وا . و ات KLF6 ٢٧ %. م ا ٣٦ % ا ت /٨( ٢٢ ). ).
آ أن ا ات ا ت ا ا آ ن اآ ن ٢ KLF6 ، آ ان ا ا ع Missence . . Transversion &
و ه ا را ا ص ان ا ا دث KLF6 ا و م ا ا ن وا ا ا ا را ا ا ور ا ح ا ا ا ل ا ل ا ن وا ه ء ا . ان س ه ا ات ا ان م آ ا ا ا ت ا ا ل ا ا ن وا . .
٢ ٢
Arabic summary
و ا ا اض ا ت ا ن وا ا وث ات KLF6 و م ا و ا ه ا را ا ام ا ة ات ر اى ل ا او ا د اى ا ل ا ل ا . .
٣ ٣