ABSTRACT

DEPARTMENT OF BIOLOGY

LANG, JIMMIE B.S. BALL STATE UNIVERSITY, 2016

THE LOCALIZATION OF ZIC2 IN HEK293T CELLS AND E006AA

PROSTATE CANCER CELL LINE

Committee Chair: Goodwin Ananaba, Ph.D.

Thesis dated May 2019

The Cancer Genomic Atlas (TGCA) states that nearly all prostate cancer is adenocarcinoma. Prostate cancer is measured on a Gleason score which indicates how likely it s that a tumor would spread. TGCA has produced data that shows that the protein

ZIC2 has increased RNA expression in prostate cancer cells that have a Gleason score of

3 or higher.

ZIC2 is primarily located in the central nervous system during development.

Preliminary data was shown that ZIC2 can be introduced to HEK cells 293T cells when constructing a plasmid containing ZIC2 and a N-terminal GFP tag. It was seen that the protein ZIC2 was localized in the mitochondria, which was a contrast to previous studies where ZIC were localized in the nucleus. It is hypothesized that ZIC2 changes localization when using a plasmid containing ZIC2.

THE LOCALIZATION OF ZIC2 PROTEIN IN HEK293T CELLS AND E006AA

PROSTATE CANCER CELL LINE

A THESIS

SUBMITTED TO THE FACULTY OF CLARK ATLANTA UNIVERSITY

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR

THE DEGREE OF MASTER OF SCIENCE

BY

JIMMIE LANG

DEPARTMENT OF BIOLOGY

ATLANTA, GEORGIA

MAY 2019

© 2019

JIMMIE LANG

All Rights Reserved

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ACKNOWLEDGEMENTS

I would like to acknowledge all of those who have assisted me through my academic journey at Clark Atlanta University including the Department of Biological

Sciences, my committee members Godwin Ananaba, Ph.D., Nathan Bowen, Ph.D., and

Bekir Cinar, Ph.D., and all members of the Bowen Lab. I would like to give a special acknowledgement to my mother, Cynthia Clayton-Marshall, who has encouraged me through this entire process, and also to my lab partner, Janelle Moore, who has not only motivated me but has made this such a meaningful journey for me.

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TABLE OF CONTENTS

ACKNOWLEDGEMENTS ...... ii

LIST OF FIGURES………………….…………………………………………………....v

CHAPTER

I. INTRODUCTION…...…………………………………………...……….…..…..1

Prostate Cancer Statistics….....…………………………………………….…...…1

Androgen Independent Prostate Cancer……………………………………..…….2

II. LITERATURE REVIEW…………………………………………………………4

Zinc Finger Proteins..………………………………………….………………….4

ZIC Proteins………………………………………………………………….……5

III. MATERIALS AND METHODS……………….…………………………………8

Transformation of Competent Cells and Extraction of Colonies………...... ….8

Isolation of Plasmid…………………………………..………………..………….9

Cell Culture……………………..……..……………………………………..……9

Transfection of E006AA and HEK 293T Cells…….……………………………..9

Protein Extraction and Western Analysis……………...…………………….……9

Confocal Imaging………………………………………...…………….………...10

IV. RESULTS………………………………………….…………………………….11

Western Analysis……………………..………………...……….…….………....11

Confocal Imaging……………………………….………………………………..12

V. CONCLUSION AND DISCUSSION…………………..……………………….13

iii

REFERENCES..…………………………………………………………………………14

iv

LIST OF FIGURES

Figure

1. Expression of ZIC2 in prostate cancer cells…………………...………………….2

2. Cys2His2 protein motif of a zinc finger…..………………………….…..……….5

3. Five human ZIC family protein with conserved regions………………………….6

4. due to a mutation in ZIC2 during development……...………7

5. Localization of ZIC2 in the mitochondria of HEK 293T cells using GFP,

Mitotracker Red, and DAPI…………………………….....………………………7

6. Origene ZIC2 ORF with a C-terminal GFP tag plasmid……….………...……….8

7. Western analysis of transient and stable transfection……………………………11

8. Confocal imaging of E006AA EGFP cells and HEK293T ZGFPC’ cells..…..….12

9. Confocal imaging of E006AA ZGFPC’ cells and E006AA ZGFPN’ cells……...12

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CHAPTER I

INTRODUCTION

Prostate Cancer Statistics

According to the American Cancer Society, cancer is the second cause of death in the United States with prostate cancer being the second leading cancer related death in men (2018). The United States Centers for Disease Control and Prevention estimated that there were 164,690 new cases of prostate cancer in 2018 accounting for 19% of cancer in all males (2018). In addition, there is an estimated 29,430 deaths from prostate cancer making up 9% of cancer related deaths in males (Siegel et al., 2018).

Because of widespread prostate-specific antigen (PSA) testing, increases in prostate cancer were seen in 1990. The antigen is produced in the normal epithelial cells of the prostate gland and it also produced by prostate cancer cells. In normal conditions the antigen is present at low levels but the levels increase when produced by prostate cancer cells (Zijstra and Stoorvogel, 2016). There has been a reduction in prostate cancer as a result of a decrease in PSA testing and Prostate cancer can be detected while they are still localized to the gland (Siegel et al., 2018). However, the problem is that PSA testing does not distinguish whether the cancer is lethal or nonlethal. The issue then arises to determine which men should be given treatment and which men do not require treatment.

One solution is tracking the PSA level over a period of years. The Baltimore

1 2 Longitudinal Aging Study determined that before a diagnosis, the PSA of men who died of prostate cancer had PSA levels that increased at an exponential rate. With concern of overdiagnosis and overtreatment, the US Preventive Service Task Force gave recommendation in April 2017 that men 55 to 69 years old should be screened for prostate cancer (Siegel et al., 2018). Prostate cancer is graded on the Gleason grading system with prostate cancers having a Gleason score of 5-7 are considered to be moderately differentiated (Figure 1).

Figure 1. Expression of Zic2 in prostate cancer cells.

The survival rate for cancer is lower for black patient than white patients for every type of cancer except kidney and pancreas. In addition, black patients are more likely to be diagnosed with cancer at a more advanced stage. The cancer occurrence and outcome varies between racial and ethnic groups due to socio-economic differences (wealth, early detection, and treatment).

Androgen Independent Prostate Cancer

Prostate cancer develops in differentiated epithelial cells where the activation of replication pathways and the inactivation of tumor suppressor allow the cancer

3 cells to grow at increased levels and survive in conditions where the cell would normally undergo apoptosis. In prostate cancer, the androgen testosterone acts as a promoter for increased growth and proliferation. When testosterone enters prostate cells or prostate cancer cells it is converted into its active form of dihydroxytestesterone

(DHT). DHT binds to the androgen receptor causing the receptor to dimerize and becomes phosphorylated. After phosphorylation the receptor travels to the nucleus and binds on the androgen response elements (Taichman et al., 2007).

The first step in treatment for prostate cancer is to stop proliferation of prostate cancer cells by chemotherapeutic androgen deprivation or castration. This causes majority of the prostate cancer cells to die. However, during the period of remission the cells that become androgen independent continue to grow and proliferate (Dillard et al.,

2008).

CHAPTER II

LITERATURE REVIEW

Zinc Finger Proteins

Zinc finger domains are protein structures stabilized by the binding of a zinc ion.

The most common type of Zinc finger proteins are identified by the Cys2His2 domain

(Figure 2). Normally, a Cys2His2 is characterized as a short protein motif that contains two cysteine and two histidine residues that bonds to a zinc ion. Most contain an alpha helix and an antiparallel beta structure (Razin et al., 2012). Zinc finger proteins are an important class of genomic regulators being found throughout the eukaryotic genome, counting for an estimated 3% of genes (Brayer and Segal, 2008). Zinc finger proteins are able to bind to DNA while also having the function to participate in protein-protein interactions (Wolfe et al., 2000). In multi-finger zinc fingers, only 3-4 zinc finger domains are involved in the DNA binding and the others are involved in other types of interactions. However, the interactions with the DNA are usually mediated by the amino acids located on the N-terminal region of the alpha helix of the zinc finger (Brayer and

Segal, 2008).

4 5

Figure 2. Cys2His2 protein motif of a Zinc Finger.

The interaction between the zinc finger and DNA is done by interacting with the hydrogen donors and acceptors in the major groove of the DNA (Razin et al., 2012). It is seen in the Ikaros family of proteins that all members of the protein family use their C- terminal fingers to mediate protein-protein interactions and the N-terminal fingers bind to

DNA (Perdomo et al., 2000). Zinc fingers that are involved with protein-protein interactions may also have additional conserved motifs that seen in the regulation of intracellular localization and expression (Razin et al., 2012).

ZIC Protein

The ZIC family proteins are zinc finger proteins defined by zinc finger domains that contain five Cys2His2 zinc fingers (Brayer and Segal, 2008). There are five members of the ZIC family proteins and each member contains a conserved region in the N- terminal domain of the zinc finger (Figure 3). The structure of the ZIC family is most closely related to zinc finger domains of other proteins including the GLI family

(Houtmeyers et al., 2013). The zinc fingers of the ZIC family protein contain a DNA binding domain and also domains that participate in protein-protein interactions (Koyabu, et al., 2001). It was seen that ZIC3 has been shown to contain a nuclear localization

6 signal that when mutated stops the transport of ZIC3 to the nucleus (Hatayama et al., 2008). Interactions between ZIC and other proteins have also been shown to alter the sub-cellular localization of the ZIC proteins and it is possible that the shuttling of the ZIC proteins between the nucleus and the cytoplasm is controlled by other interacting proteins

(Zijlstra and Stoorvogel, 2016).

Figure 3. Five human ZIC family protein with conserved regions. The zinc finger regions are shown by the blue circles. ZIC1, 2 and 3 all shared the same 5 five conserved zinc finger regions. There is also another conserved region (ZOC) located near the N- terminal end of ZIC 1, 2 and 3 that is a conserved sequence in each protein.

The ZIC family zinc finger proteins play an important role in development. ZIC2 plays a role in neural development and it was discovered that mutations in ZIC2 caused holoprosencephaly (Figure 4), a malformation of the proper formation of the structures of the forebrain in particular the left-right asymmetry and the development of the central nervous system. (Brown, 2001). In a quantitative study done on ovarian cancer, there were 61 genes that were expressed at a high level in malignant form tumors. Among

7 those genes was ZIC2 (Marchini et al., 2012). ZIC2 was also seen overexpressed in endometrial cancer suggesting that it may be associated with cancers of the bladder, colon, and breast.

Figure 4. Holoprosencephaly due to a mutation in ZIC2 during development.

The preliminary data has shown that ZIC2 can be introduced into a HEK 293T cell using a plasmid containing a GFP tag located on the amino-end of the protein

(Bylund et al., 2004) (Figure 5). We hypothesized that moving the GFP tag to the carboxyl end of the ZIC2 protein would change localization of the ZIC2 protein (Zhang and Crandall, 2007). To test the hypothesis, the following aims were investigated:

1. To determine the expression of ZIC2 in E006AA and HEK 293T cells. 2. To determine the effects of the GFP tag located on the Carboxyl end of the ZIC protein.

A B C D

Figure 5. Localization of Zic2 in the mitochondria of HEK 293T cells using GFP(A), Mitotracker Red(B) and DAPI(C). These images collectively show that the GFP was localized to the mitochondria in the cells (D).

CHAPTER III

MATERIALS AND METHODS

Transformation of Competent Cells and Extraction of Colonies

Competent E. coli cells were purchased from Agilent Technologies and transformed with an Origene ZIC2 ORF with a C-terminal GFP tag plasmid (Figure 6).

The pUC18 plasmid was used as a control. The E. coli were grown on LB agar plates containing ampicillin. Single colonies grown on the agar plates were then extracted and grown in LB broth overnight.

Figure 6. Origene ZIC2 ORF with a C-terminal GFP tag plasmid

8 9 Isolation of Plasmid

Using the Qiagen plasmid mini kit, the ZIC2 plasmid was extracted from the

E.coli and the concentration of the plasmid was measured using a nanodrop spectrophotometer. The Zymopure II plasmid maxiprep kit was used to increase the plasmid concentration and the concentration was measured again using the nanodrop spectrophotometer.

Cell Culture

E006AA cells were purchased from Roswell Park Cancer Institution

(Koochekpour et al., 2004) and HEK 293T cells were purchased from ATCC. The cells were grown in Dulbecco’s Mod of Eagle’s Medium with L-glutamine, 10% Fetal Bovine

Serum, and 1% penstrep. The cells were incubated at 37 C. Cells were passaged to new plates with 1x PBS solution with gentamicin sulfate (50mg/ml) and treated with 1x trypsin with EDTA.

Transfection of E006AA and HEK 293T cells

For the first transfection the cells were grown in 1x Opti-MEM containing

HEPES, Sodium Bicarbonate, L-glutamine. They were treated with Promega Fugene HD and selected by the use of G418 Sulfate (50mg/mL). The second transfection was done the same using Promega Fugene.

Protein Extraction and Western Analysis

The protein extraction was done by lysing the cell using Lammeli with 5%

Betamercaptoethanol (BME). A western analysis was done using Genscript Transfer

Buffer Powder and Genscript Running Buffer. The blot was stained with Amresco

Ponceau Stain. The primary antibody was an ABCAM Anti-ZIC2 antibody and the

10 Secondary Antibody was a Goat Anti-Rabbit IgG HRP conjugate. The blot was washed with TBST 1x with Tween 20 and 5% milk in TBST.

Confocal Imaging

To prepare cells for imaging using the Zeiss Confocal Microscope, the cells were stained with Invitrogen Mitotracker and LifeTechnologies Molecular Probe Nuc Blue

Fixed cell stain DAPI.

CHAPTER IV

RESULTS

Western Analysis

Western Analysis was done to show determine the expression of ZIC2 in E006AA cells and HEK293T cells (Figure 7). The anit-ZIC2 antibody detects ZIC2 65kDa. The beta actin analysis was done to observe loading technique. The bands on both the transient and stable transfection show that the ZIC2 protein was present in all cell lines.

Transient Transfection

Stable Transfection

’

’

’

PR

HEK

EGFP

ZGFPC

ZGFPC

E006AA E006AA

E006AA E006AA

ZGFPN

E006AA E006AA E006AA E006AA

HEK HEK E006AA E006AA E006AA EGFP ZGFPC’ EGFP ZGFPN’ ZGFPC’

Beta Actin Beta Actin

Figure 7. Western Analysis of transient and stable transfection. ZIC2 protein observed at 65kDa. Beta Actin to observe loading technique.

11 12 Confocal Imaging

Confocal imaging was done of the E006AA cells and the HEK293T cells (Figures

8 and 9). The localization of the ZIC2 protein was determined by the green fluorescence protein. It was observed that the GFP was overlapped with the mitotracker red. The nucleus of each cells was stained blue with the DAPI.

E006AA EGFP HEK293T ZGFPC’

A B A B

C D C D

Figure 8. Confocal imaging of E006AA EGFP cells and HEK293T ZGFPC’ cells. (A) GFP (B) Mitotracker Red (C) DAPI (D) GFP, Mitotracker Red and DAPI combined. E006AA ZGFPC’ E006AA ZGFPN’

A B A B

C D C D

Figure 9. Confocal imaging of E006AA ZGFPC’ cells and E006AA ZGFPN’ cells. (A) GFP (B) Mitotracker Red (C) DAPI (D) GFP, Mitotracker Red and DAPI combined.

CHAPTER V

CONCLUSION AND DISCUSSION

By studying the localization of ZIC2, it brings the attention to the need to study the insertion of a GFP tag and the effects it can have on the protein properties. The results suggest that the ZIC2 protein is localized in the mitochondria. The overlapping of the

GFP and the mitrotracker red provides evidence for the localization. In previous studies, the mitochondria seems to play a role in providing a resistance to apoptosis in African

American men with prostate cancer (Chaudhary et al., 2016). In addition, mitochondrial dysfunction has been seen to cause resistance against other conventional anticancer regimens in tumors (Chaudhary et al., 2016). Mutations during any process of the glycolytic pathway can cause mitochondrial dysfunction and promote tumor growth

(Corbet and Feron, 2017). This provides insight on the localization of ZIC2 protein in cancer cell lines and shows evidence that further investigation should be done to determine the function of the ZIC2 protein in the mitochondria of cancer cells.

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