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ONCOGENOMICS Deaths in the United States and Other Developed 2000) Oncogene (2002) 21, 3814 ± 3825 ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc Identi®cation of genes over-expressed in small cell lung carcinoma using suppression subtractive hybridization and cDNA microarray expression analysis Chaitanya S Bangur*,1, Ann Switzer1, Liqun Fan1, Matthew J Marton2, Michael R Meyer2 and Tongtong Wang1 1Tumor Antigen Discovery, Corixa Corporation, 1124 Columbia Street, Seattle, Washington WA 98104, USA; 2Rosetta Inpharmatics, 12040 115th Avenue NE, Kirkland, Washington, WA 98034, USA To identify genes that are dierentially over-expressed in rate for patients diagnosed with NSCLC is 10 ± 15%, Small Cell Lung Carcinoma (SCLC) we have used a while patients diagnosed with SCLC have an even combination of suppression subtractive hybridization and worse prognosis with a 5-year survival rate of 55% cDNA microarray to analyse the expression pro®les of (Ginsberg et al., 1997; Ihde et al., 1997). Although, 2400 cDNAs clones. Genes that are over-expressed in SCLC is initially highly responsive to radiation and SCLC were identi®ed using 32 pairs of ¯uorescence- chemotherapy (50 ± 90%), most patients tend to relapse labeled cDNA samples representing various lung tumors with highly resistant disease within a year of treatment and normal tissues. This comprehensive approach has (Ihde et al., 1997). Most patients diagnosed with SCLC resulted in the identi®cation of 209 genes that are have regional lymph-node involvement or disseminated dierentially over-expressed in SCLC. Quantitative real- disease at the time of initial diagnosis (Ihde, 1992), time PCR was used to further validate the expression of which strongly correlates with the poor prognosis 43 genes in SCLC tumors and various normal tissues. observed for these patients. Thus, the problem with Discussed in this report are nine genes, which showed the the management of lung cancer can be attributed to the most promising SCLC tumor to normal tissue dierential lack of reliable early detection methods and the expression pro®les, including seven known and two novel inadequacies of current treatment protocols. This has genes. The large number of dierentially expressed genes warranted the eorts towards identi®cation of new identi®ed from this analysis and the characterization of molecular targets for designing more reliable diagnostic these genes will provide valuable information in better methods and novel therapeutic protocols for the understanding the biology of SCLC and help us in treatment of this malignancy. developing these gene products as potential targets for SCLC tumors are distinguished by their neuroendo- diagnostic as well as therapeutic usage. crine phenotype and frequently stain for neuroendo- Oncogene (2002) 21, 3814 ± 3825. DOI: 10.1038/sj/ crine markers such as neuron-speci®c enolase, onc/1205480 chromogranin, and synsptophysin (Guinee et al., 1994). SCLC cells are also found to secrete several Keywords: small cell lung carcinoma (SCLC); micro- autocrine growth factors and hormones including array; subtractive hybridization; real-time PCR; gene gastrin-releasing peptide, neurotensine, vasopressin, expression and cholecystokinin (Kalemkerian, 2000; Williams, 1997). Some of the autocrine growth factors have been used as prognostic markers of this disease with varying Introduction levels of success and reliability (Feld et al., 2000). However, their use as diagnostic and therapeutic Lung cancer is the most common cause of cancer targets has not been very successful (Kalemkerian, ONCOGENOMICS deaths in the United States and other developed 2000). Thus the need of new reliable and eective countries. It accounts for approximately 30% of all targets for the early detection and management of this cancer deaths and in the United States alone 4150 000 malignancy is more than ever. It has been well people die of lung cancer every year (Parker et al., documented that cancer cells go through a lot of 1997). Lung tumors are divided into two major types, genetic and epigenetic changes (Lengauer et al., 1998; NSCLC and SCLC. 80% of lung cancer incidences are Williams, 1997), which is suggested to bring about attributed to the NSCLC type, while SCLC accounts dramatic changes in the gene expression pro®les of for the rest 20% of cases. The overall 5-year survival these cells. The past decade has seen the development of several technologies for pro®ling these changes in gene expression, including dierential and subtractive hybridization, dierential display, SAGE, and micro- *Correspondence: CS Bangur; E-mail: [email protected] Received 11 January 2002; revised 6 March 2002; accepted 18 arrays (Gray and Collins, 2000). These approaches March 2003 have proven to be extremely useful in providing a SCLC gene expression profiling CS Bangur et al 3815 comprehensive look at the biology of various cancers as well as in the identi®cation of new targets for the development of novel therapeutic approaches for the treatment of these cancers (Backert et al., 1999; Nacht et al., 1999; Nocito et al., 2001; Ono et al., 2000; Sgroi et al., 1999; Wang et al., 2000; Xu et al., 2000; Yang et al., 1999). Recent studies have documented a large number of genetic alterations present in SCLC (Girard Figure 1 Subtraction eciency as determined by depletion of et al., 2000; Wistuba et al., 2000a,b), which is GAPDH from the subtracted cDNA population. Equal amounts indicative of possible changes in the gene expression of subtracted and unsubtracted cDNAs were subjected to PCR pro®le. However, little has been done in exploring ampli®cation for the indicated number of cycles using GAPDH these changes in SCLC and the only study reported to gene speci®c primers. PCR products were run on a 1% agarose gel and stained with ethidium bromide. M, molecular weight date was very limited in scope and was designed to marker address the dierences between the SCLC subtypes at the molecular level (Anbazhagan et al., 1999). This study was undertaken to carryout a compre- hensive analysis of the changes in gene expression in unsubtracted cDNAs, GAPDH speci®c PCR product SCLC and was speci®cally focused towards identi®ca- is visible by the 23rd round of ampli®cation. However, tion of genes that are dierentially over-expressed in in case of the subtracted cDNAs, the GAPDH speci®c SCLC. To accomplish this we used a combination of PCR product is visible only after 33 rounds of technologies including suppression subtractive hybridi- ampli®cation. This is indicative of preferential deple- zation (SSH) for cDNA subtraction (Diatchenko et al., tion of GAPDH and most ubiquitously expressed 1996), high-density cDNA microarray and quantitative genes from the subtracted cDNA mixture. Sequence real-time RT ± PCR. Several recent reports have shown analysis of 48 ± 96 randomly picked clones gave us a the successful use of similar technologies in identifying good estimation of the complexity of the subtracted novel and previously unknown genes found to be libraries. Generally the libraries generated by the dierentially expressed in breast, prostate, head and suppression subtractive hybridization method are of neck, and lung squamouscell carcinoma (Villaret et al., high complexity because of the normalization of highly 2000; Wang et al., 2000; Xu et al., 2000; Yang et al., abundant messages during hybridization. However, in 1999). Here we report the identi®cation of 209 genes case of the SCL2 subtracted cDNA library, we that are dierentially over-expressed in SCLC com- repeatedly recovered cDNAs for GRP and ASH1, pared to normal tissues and the initial characterization which are known to be over-expressed in SCLC (Ball et of nine of these genes. Further characterization of these al., 1993; Yamaguchi et al., 1983). Approximately 30% nine genes and other genes identi®ed in this study will of the clones from this library contained cDNA inserts be helpful in de®ning a panel of diagnostic and representing either of the two genes (data not shown). therapeutic targets for SCLC and better our under- The repeated recovery of these genes was an indication standing into the biology of this malignancy. of successful subtraction for enriching genes dieren- tially expressed in SCLC. However, it also meant that the SCL2 library had a set of highly redundant clones and was not complex enough to be screened further by Results microarray. To overcome this problem, two additional subtracted libraries, SCL3 and SCL4, were constructed Generation and characterization of SCLC specific cDNA using a driver cDNA pool for hybridization that libraries included the cDNAs for GRP and ASH1 in addition To enrich for genes preferentially expressed in SCLC, to the nine normal tissues used for the SCL2 library we generated four SCLC tumor speci®c cDNA libraries (see Materials and methods). The inclusion of the GRP using the suppression subtractive hybridization method and ASH1 cDNAs in the driver pool decreased the (Diatchenko et al., 1996). These subtracted libraries are percentage of clones that represented these two genes referred to as SCL1, SCL2, SCL3 and SCL4 (for to around 5% in the SCL3 and SCL4 libraries. Thus details, see materials and methods). The initial by spiking in the cDNAs for the two highly abundant characterization of these subtracted libraries was done genes we were able to generate libraries that were of by estimating the eciency of subtraction and sucient complexity that could be exploited further by sequencing of 48 ± 96 randomly picked clones from cDNA microarray analysis. each library. The eciency of subtraction was determined by comparing the abundance of glycer- cDNA microarray analysis aldehyde-3-phosphate dehydrogenase (GAPDH) cDNAs before and after subtraction. GAPDH is a Twenty-four thousand randomly selected cDNA clones good representative for constitutively expressed genes, from SCL1, SCL3, and SCL4 subtracted libraries were which are highly abundant in most tissues. Figure 1 PCR ampli®ed and arrayed onto 32 replicate glass shows the depletion of GAPDH from the subtracted slides (microarrays).
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