The connection between E2F and ATM in p53-dependent apoptosis

Programmed cell death is an anticancer mechanism utilized by p53 that when disrupted can accelerate tumor development in response to oncogenic stress. Defects in the RB tumor suppressor cause aberrant cell proliferation as well as apoptosis. The combinatorial loss of the p53 and RB pathways is observed in a large percentage of human tumors. The E2F family of transcription factors primarily mediates the phenotype of Rb loss, since RB is a negative regulator of E2F. Contrary to early expectations, it has now been shown that the ARF (alternative reading frame) tumor suppressor is not required for p53-dependent apoptosis in response to deregulation of the RB/E2F pathway. In this study, we demonstrate that ATM, known as a DNA double-strand break (DSB) sensor, is responsible for ARF-independent apoptosis and p53 activation induced by deregulated E2F1. Moreover, NBS1, a component of the MRN DNA repair complex, is also required for E2F1-induced apoptosis and apparently works in the same pathway as ATM. We further found that endogenous E2F1 and E2F3 both play a role in apoptosis and ATM activation in response to inhibition of RB by the adenoviral E1A oncoprotein. We demonstrate that, unlike deregulated E2F3 and Myc, ATM activation by deregulated E2F1 does not involve the induction of DNA damage, autophosphorylation of ATM on Ser 1981, a marker of ATM activation by DSB, but does depend on the presence of NBS1, suggesting that E2F1 activates ATM in a different manner from E2F3 and Myc. Results from domain mapping studies show that the DNA binding, dimerization, and marked box domains of E2F1 are required to activate ATM and stimulate apoptosis but the transactivation domain is not. This implies that E2F1's DNA binding and interaction with other proteins through the marked box domain are necessary to induce ATM activation leading to apoptosis but transcriptional activation by E2F1 is dispensable. Together these data suggest a model in which E2F1 activates ATM to phosphorylate p53 through a novel mechanism that is independent of DNA damage and transcriptional activation by E2F1.^

Impact of COP9 signalosome subunit 6 (CSN6) on MDM2-p53 axis in DNA damage-mediated apoptosis and tumorigenesis

Mammalian COP9 signalosome, which connects signaling with the ubiquitin-mediated proteasome degradation pathway, is implicated in cell cycle regulation and DNA damage response. However, whether COP9 is dysregulated in cancers has not been well established. Here, we showed that COP9 subunit 6 (CSN6) was upregulated in malignant breast and thyroid tumors and positively correlated with MDM2 expression. Investigation of the underlying mechanism suggested that CSN6 stabilized MDM2, thereby accelerating the degradation of p53. We generated mice carrying a targeted disruption of the Csn6 gene, and found that the mice with both alleles disrupted (Csn6-/- ) died in early embryogenesis (E7.5). Csn6+/- mice were sensitized to undergo γ-radiation-induced p53-dependent apoptosis in both thymus and developing central nervous system. Consequently. Csn6 +/- mice were more susceptible to the lethal effects of high-dose γ-radiation than wild-type mice. Notably, Csn6+/- mice were less susceptible to γ-radiation-induced tumorigenesis and had better long-term survival after low-dose γ-radiation exposure compared with wild-type animals, indicating that loss of CSN6 enhanced p53-mediated tumor suppression in vivo. In summary, the regulation of MDM2-p53 signaling by CSN6 plays a significant role in DNA damage-mediated apoptosis and tumorigenesis, which suggests that CSN6 may potentially be a valuable diagnostic marker for cancers with a dysregulated MDM2-p53 axis. ^

Role of telomere dysfunction, DNA damage response and p53 mutations in tumorigenesis and aging

The ends of eukaryotic chromosomes are protected by specialized ribonucleoprotein structures termed telomeres. Telomeres protect chromosomes from end-to-end fusions, inappropriate repair and degradation. Disruption of this complex activates an ATM/ATR DNA damage response (DDR) pathway. One component of the complex is the Protection Of Telomeres 1 (POT1) protein, an evolutionarily conserved protein which binds single-stranded 3' overhang and is required for both chromosomal end protection and telomere length regulation. The mouse contains two POT1 orthologs, Pot1a and Pot1b. Here we show that both proteins colocalize with telomeres through interaction with the adapter protein TPP1. In addition, compared to Pot1a, the OB-folds of Pot1b possess less sequence specificity for telomeres. Disruption of POT1 proteins result in telomere dysfunction and activation of an ATR-dependent DDR at telomeres, suggesting that this response is normally suppressed by POT1 binding to the single-stranded G-overhang. ^ Telomeres are maintained by telomerase, and its absence in somatic cells results in telomere progressive loss that triggers the activation of p53. Telomere dysfunction initiates genomic instability and induces both p53-dependent replicative senescence and apoptosis to suppress tumorigenesis. In the absence of functional p53, this genomic instability promotes cancer. It was previously not known which aspect of the p53 dependent DNA damage response is important to suppress tumorigenesis initiated by dysfunctional telomeres. The p53R172P knock-in mouse, which is unable to induce apoptosis but retains intact cell cycle arrest/cellular senescence pathways, allowed us to examine whether p53-dependent apoptosis is a major tumor suppression pathway initiated in the setting of telomere dysfunction. Spontaneous tumorigenesis remains potently suppressed in late generation telomerase null mice possessing the p53P/P mutation. These results suggest that suppression of spontaneous tumorigenesis initiated by dysfunctional telomeres requires activation of a p53-dependent senescence pathway. In addition, we used another knock-in mouse model with a p53R172H (p53H) point mutation to test the hypothesis that telomere dysfunction promotes chromosomal instability and accelerates the onset of tumorigenesis in vivo in the setting of this most common gain-of-function mutation in the human Li Fraumeni cancer syndrome. We unexpectedly observed that telomerase null mice possessing dysfunctional telomeres in the setting of the p53H/+ mutation develop significantly fewer tumors, die prematurely and exhibit higher level of cellular senescence, apoptosis and elevated genomic instability compared to telomerase intact p53H/+ and telomerase null p53+/+ mice. These contrasting results thus link cancer and aging to the functional status of telomeres and the integrity of the p53 pathway. ^

Molecular mechanisms by which p53/LSD1 and ERalpha/Trim24 complexes mediate gene regulation

In this dissertation, I identify two molecular mechanisms by which transcription factors cooperate with their co-regulators to mediate gene regulation. In the first part, I demonstrate that p53 directly recruits LSD1, a histone demethylase, to AFP chromatin to demethylate methylated H3K4 and actively mediate transcription repression. Loss of p53 and LSD1 interaction at chromatin leads to derepression of AFP in hepatic cells. In the second part, I reveal that Trim24 functions as an important co-activator in ERα-mediated gene activation in response to estrogen stimulation. Trim24 is recruited by ligand-bound ERα to chromatin and stabilizes ERα-chromatin interactions by binding to histone H3 via its PHD finger, which preferentially recognizes unmethylated H3K4. ^

DNA methylation inhibits p53 mediated survivin repression in cancer cells

Survivin (BIRC5) is a member of the Inhibitor of Apoptosis (IAP) gene family and functions as a chromosomal passenger protein as well as a mediator of cell survival. Survivin is widely expressed during embryonic development then becomes transcriptionally silent in most highly differentiated adult tissues. It is also overexpressed in virtually every type of tumor. The survivin promoter contains a canonical CpG island that has been described as epigenetically regulated by DNA methylation. We observed that survivin is overexpressed in high grade, poorly differentiated endometrial tumors, and we hypothesized that DNA hypomethylation could explain this expression pattern. Surprisingly, methylation specific PCR and bisulfite pyrosequencing analysis showed that survivin was hypermethylated in endometrial tumors and that this hypermethylation correlated with increased survivin expression. We proposed that methylation could activate survivin expression by inhibit the binding of a transcriptional repressor. ^ The tumor suppressor protein p53 is a well documented transcriptional repressor of survivin and examination of the survivin promoter showed that the p53 binding site contains 3 CpG sites which often become methylated in endometrial tumors. To determine if methylation regulates survivin expression, we treated HCT116 cells with decitabine, a demethylation agent, and observed that survivin transcript and protein levels were significantly repressed following demethylation in a p53 dependent manner. Subsequent binding studies confirmed that DNA methylation inhibited the binding of p53 protein to its binding site in the survivin promoter. ^ We are the first to report this novel mechanism of epigenetic regulation of survivin. We also conducted microarray analysis which showed that many other cancer relevant genes may also be regulated in this manner. While demethylation agents are traditionally thought to inhibit cancer cell growth by reactivating tumor suppressors, our results indicate that an additional important mechanism is to decrease the expression of oncogenes. ^

Regulation of estrogen receptor alpha by the tumor suppressor p53 in breast cancer cells

Estrogen receptor (ER) and the tumor suppressor p53 are key prognostic indicators in breast cancer. Estrogen signaling through its receptor (ER) controls proliferation of normal as well as transformed mammary epithelial cells, and the presence of ER is established as a marker of good prognosis and response to therapy. The p53 tumor suppressor gene is often referred to as the "cellular gatekeeper" due to its extensive control of cell proliferation and apoptosis. Loss of functional p53 is a negative prognostic indicator and is correlated with lack of response to antiestrogens, reduced disease-free interval and increased chance of disease recurrence. Clinical studies have demonstrated that tumors with mutated p53 tend to be ER negative, while ER positive tumors tend to have wild type p53. ^ Recent studies from our lab indicate that p53 genotype correlates with estrogen receptor expression in mammary tumors in vivo. We therefore hypothesized that p53 regulates ER expression in mammary cancer cells by recruitment of specific cofactors to the ER promoter. To test this, MCF-7 cells were treated with doxorubicin or ionizing radiation, both of which stimulated significant increases in p53 expression, as expected, but also increased ER expression in a p53-dependent manner. Furthermore, in cells treated with siRNA targeting p53, both p53 and ER protein levels were significantly reduced. P53 was also demonstrated to transcriptionally regulate the ER promoter in luciferase assays and chromatin immunoprecipitation assays showed that p53 was recruited to the ER promoter along with CARM1, CBP, c-Jun and Sp1 and that this multifactor complex was formed in a p53-dependent manner. The regulation of ER by p53 has therapeutic implications, as the treatment of breast cancer cells with doxorubicin sensitized these cells to tamoxifen treatment. Furthermore, response to tamoxifen as well as to estrogen was dependent on p53 expression in ER positive human breast cancer cells. Taken together, these data demonstrate that p53 regulates ER expression through transcriptional control of the ER promoter, accounting for their concordant expression in human breast cancer and identifying potentially beneficial therapeutic strategies for the treatment of ER positive breast cancers. ^

Genetic epidemiologic methods in risk determination in Li-Fraumeni syndrome

Li-Fraumeni syndrome (LFS) is characterized by a variety of neoplasms occurring at a young age with an apparent autosomal dominant transmission. Individuals in pedigrees with LFS have high incidence of second malignancies. Recently LFS has been found to be associated with germline mutations of a tumor-suppressor gene, p53. Because LFS is rare and indeed not a clear-cut disease, it is not known whether all cases of LFS are attributable to p53 germline mutations and how p53 plays in cancer occurrence in such cancer syndrome families. In the present study, DNAs from constitutive cells of two-hundred and thirty-three family members from ten extended pedigrees were screened for p53 mutations. Six out of the ten LFS families had germline mutations at the p53 locus, including point and deletion mutations. In these six families, 55 out of 146 members were carriers of p53 mutations. Except one, all mutations occurred in exons 5 to 8 (i.e., the "hot spot" region) of the p53 gene. The age-specific penetrance of cancer was estimated after the genotype for each family member at risk was determined. The penetrance was 0.15, 0.29, 0.35, 0.77, and 0.91 by 20, 30, 40, 50 and 60 year-old, respectively, in male carriers; 0.19, 0.44, 0.76, and 0.90 by 20, 30, 40, and 50 year-old, respectively, in female carriers. These results indicated that one cannot escape from tumorigenesis if one inherits a p53 mutant allele; at least ninety percent of p53 carriers will develop cancer by the age of 60. To evaluate the possible bias due to the unexamined blood-relatives in LFS families, I performed a simulation analysis in which a p53 genotype was assigned to each unexamined person based on his cancer status and liability to cancer. The results showed that the penetrance estimates were not biased by the unexamined relatives. I also determined the sex, site, and age-specific penetrance of breast cancer in female carriers and lung cancer in male carriers. The penetrance of breast cancer in female carriers was 0.81 by age 45; the penetrance of lung cancer in male carriers was 0.78 by age 60, indicating that p53 play a key role for tumorigenesis in common cancers. ^

CHARACTERIZATION OF TCL1-Tg:P53-/- MICE THAT RESEMBLE HUMAN CHRONIC LYMPHOCYTIC LEUKEMIA WITH 17P-DELETION

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the United Statesand Europe. CLL patients with deletion of chromosome 17p, where the tumor suppressor p53 gene is located, often develop a more aggressive disease with poor clinical outcomes. However, the underlying mechanism remains unclear. In order to understand the underneath mechanism in vivo, I have recently generated mice with Eu-TCL1-Tg:p53-/- genotype and showed that these mice develop aggressive leukemia that resembles human CLL with 17p deletion. The Eu-TCL1-Tg:p53-/- mice developed CLL disease at 3-4 months, significantly earlier than the parental Eu-TCL1-Tg mice that developed CLL disease at 8-12 months. Flow cytometry analysis showed that the CD5+/ IgM+ cell population appeared in the peritoneal cavity, bone marrow, and the spleens of Eu-TCL1-Tg:p53-/- mice significantly earlier than that of the parental Eu-TCL1-Tg mice. Massive infiltration and accumulation of leukemia cells were found in the spleen and peritoneal cavity. In vitro study showed that the leukemia cells isolated from the Eu-TCL1-Tg:p53-/- mice were more resistant to fludarabine treatment than the leukemia cells isolated from spleens of Eu-TCL1-Tg mice. Interestingly, TUNEL assay revealed that there was higher apoptotic cell death found in the Eu-TCL1-Tg spleen tissue compared to the spleens of the Eu-TCL1-Tg:p53-/- mice, suggesting that the loss of p53 compromises the apoptotic process in vivo, and this might in part explain the drug resistant phenotype of CLL cells with 17p-deletion. In the present study, we further demonstrated that the p53 deficiency in the TCL1 transgenic mice resulted in significant down-regulation of microRNAs miR-15a and miR16-1, associated with a substantial up-regulation of Mcl-1, suggesting that the p53-miR15a/16-Mcl-1 axis may play an important role in CLL pathogenesis. Interestingly, we also found that loss of p53 resulted in a significant decrease in expression of the miR-30 family especially miR-30d in leukemia lymphocytes from the Eu-TCL1-Tg:p53-/- mice. Such down-regulation of those microRNAs and up-regulation of Mcl-1 were also found in primary leukemia cells from CLL patients with 17p deletion. To further exam the biological significance of decrease in the miR-30 family in CLL, we investigated the potential involvement of EZH2 (enhancer of zeste homolog 2), a component of the Polycomb repressive complex known to be a downstream target of miR-30d and plays a role in disease progression in several solid cancers. RT-PCR and western blot analyses showed that both EZH2 mRNA transcript and protein levels were significantly increased in the lymphocytes of Eu-TCL1-Tg:p53-/- mice relative to Eu-TCL1-Tg mice. Exposure of leukemia cells isolated from Eu-TCL1-Tg:p53-/- mice to the EZH2 inhibitor 3-deazaneplanocin (DZNep) led to induction of apoptosis, suggesting EZH2 may play a role in promoting CLL cell survival and this may contribute to the aggressive phenotype of CLL with loss of p53. Our study has created a novel CLL mouse model, and suggests that the p53/miR15a/16-Mcl-1 axis & p53/miR30d-EZH2 may contribute to the aggressive phenotype and drug resistance in CLL cells with loss of p53.

STUDYING AGGREGATE FORMATION BY AMYOTROPHIC LATERAL SCLEROSIS-ASSOCIATED MUTANT SOD1 PROTEIN IN DROSOPHILA MODEL

A common pathological hallmark of most neurodegenerative disorders is the presence of protein aggregates in the brain. Understanding the regulation of aggregate formation is thus important for elucidating disease pathogenic mechanisms and finding effective preventive avenues and cures. Amyotrophic Lateral Sclerosis (ALS), also known as Lou Gehrig’s disease, is a selective neurodegenerative disorder predominantly affecting motor neurons. The majority of ALS cases are sporadic, however, mutations in superoxide dismutase 1 (SOD1) are responsible for about 20% of familial ALS (fALS). Mutated SOD1 proteins are prone to misfold and form protein aggregates, thus representing a good candidate for studying aggregate formation. The long-term goal of this project is to identify regulators of aggregate formation by mutant SOD1 and other ALS-associated disease proteins. The specific aim of this thesis project is to assess the possibility of using the well-established Drosophila model system to study aggregation by human SOD1 (hSOD1) mutants. To this end, using wild type and the three mutant hSOD1 (A4V, G85R and G93A) most commonly found among fALS, I have generated 16 different SOD1 constructs containing either eGFP or mCherry in-frame fluorescent reporters, established and tested both cell- and animal-based Drosophila hSOD1 models. The experimental strategy allows for clear visualization of ectopic hSOD1 expression as well as versatile co-expression schemes to fully investigate protein aggregation specifically by mutant hSOD1. I have performed pilot cell-transfection experiments and verified induced expression of hSOD1 proteins. Using several tissue- or cell type-specific Gal4 lines, I have confirmed the proper expression of hSOD1 from established transgenic fly lines. Interestingly, in both Drosophila S2 cells and different fly tissues including the eye and motor neurons, robust aggregate formation by either wild type or mutant hSOD1 proteins was not observed. These preliminary observations suggest that Drosophila might not be a good experimental organism to study aggregation and toxicity of mutant hSOD1 protein. Nevertheless this preliminary conclusion implies the potential existence of a potent protective mechanism against mutant hSOD1 aggregation and toxicity in Drosophila. Thus, results from my SOD1-ALS project in Drosophila will help future studies on how to best employ this classic model organism to study ALS and other human brain degenerative diseases.

Induction of synthetic lethality in mutant KRAS cells for non-small cell lung cancers chemoprevention and therapy

Lung cancer is the leading cause of cancer death in both men and women in the United States and worldwide. Despite improvement in treatment strategies, the 5-year survival rate of lung cancer patients remains low. Thus, effective chemoprevention and treatment approaches are sorely needed. Mutations and activation of KRAS occur frequently in tobacco users and the early stage of development of non-small cell lung cancers (NSCLC). So they are thought to be the primary driver for lung carcinogenesis. My work showed that KRAS mutations and activations modulated the expression of TNF-related apoptosis-inducing ligand (TRAIL) receptors by up-regulating death receptors and down-regulating decoy receptors. In addition, we showed that KRAS suppresses cellular FADD-like IL-1β-converting enzyme (FLICE)-like inhibitory protein (c-FLIP) expression through activation of ERK/MAPK-mediated activation of c-MYC which means the mutant KRAS cells could be specifically targeted via TRAIL induced apoptosis. The expression level of Inhibitors of Apoptosis Proteins (IAPs) in mutant KRAS cells is usually high which could be overcome by the second mitochondria-derived activator of caspases (Smac) mimetic. So the combination of TRAIL and Smac mimetic induced the synthetic lethal reaction specifically in the mutant-KRAS cells but not in normal lung cells and wild-type KRAS lung cancer cells. Therefore, a synthetic lethal interaction among TRAIL, Smac mimetic and KRAS mutations could be used as an approach for chemoprevention and treatment of NSCLC with KRAS mutations. Further data in animal experiments showed that short-term, intermittent treatment with TRAIL and Smac mimetic induced apoptosis in mutant KRAS cells and reduced tumor burden in a KRAS-induced pre-malignancy model and mutant KRAS NSCLC xenograft models. These results show the great potential benefit of a selective therapeutic approach for the chemoprevention and treatment of NSCLC with KRAS mutations.

Expression and function of $\beta$-1,4-galactosyltransferase during wild-type and T/{\it t\/} -mutant spermatogenesis

In the mouse, gamete recognition is mediated in part by the binding of sperm surface $\beta$1,4 galactosyltransferase (GalTase) to specific oligosaccharide residues on the zona pellucida ZP3. The expression of GalTase on the sperm surface is regulated by alleles within the distal segment of the T/t complex and results in a haploid-specific increase in GalTase expression on spermatids and sperm from t-bearing males, suggesting that differences in sperm GalTase activity may contribute to t-sperm transmission ratio distortion. In this study, the expression of GalTase RNA during wild-type and T/t-mutant spermatogenesis was characterized and the role of GalTase was analyzed in transmission ratio distortion. It was found that spermatogenic cells predominantly express the long form of the GalTase RNA, which encodes the GalTase protein that is preferentially targeted to the cell surface in somatic cells. In wild-type testes, GalTase RNA accumulates during the maturation of primary spermatocytes, reaches peak levels prior to meiosis, and decreases and meiosis. GalTase RNA accumulates to similar levels during the maturation of +/t and t/t primary spermatocytes, but unlike wild-type, the level of GalTase RNA in t-spermatocytes remains elevated during meiotic division. Consequently, spermatids in t-mutant testes inherit higher levels of GalTase RNA than do wild-type spermatids, which likely accounts for the haploid-specific increase in surface GalTase activity characteristic of spermatids from t-bearing mice.^ The functional significance of the increased GalTase activity during t-sperm transmission ratio distortion was determined by examining the distribution of GalTase RNA and surface GalTase protein in haploid spermatids from +/t males. Results show that +- and t-spermatids have similar levels of both GalTase RNA and protein, indicating that transmission ratio distortion in +/t mice is not likely due to haploid-specific differences in sperm surface GalTase activity.^ The presence of GalTase on the surface of an early spermatogenic cells before it is required on the mature sperm to perform its function during gamete binding suggests a separate function for GalTase in Sertoli-germ cell adhesion. Studies indicate that cell surface GalTase partly mediates the initial adhesion of pachytene spermatocytes, but not haploid spermatids, to Sertoli cells. ^

Expression of p53, {\it * ras\/} and PCNA in human colonic neoplasms: Possible biomarkers of malignant potential

Colorectal cancer is a leading cause of cancer mortality and early detection can significantly improve the clinical outcome. Most colorectal cancers arise from benign neoplastic lesions recognized as adenomas. Only a small percentage of all adenomas will become malignant. Thus, there is a need to identify specific markers of malignant potential. Studies at the molecular level have demonstrated an accumulation of genetic alterations, some hereditary but for the most occurring in somatic cells. The most common are the activation of ras, an oncogene involved in signal transduction, and the inactivation of p53, a tumor suppressor gene implicated in cell cycle regulation. In this study, 38 carcinomas, 95 adenomas and 20 benign polyps were analyzed by immunohistochemistry for the abnormal expression of p53 and ras proteins. An index of cellular proliferation was also measured by labeling with PCNA. A general overexpression of p53 was immunodetected in 66% of the carcinomas, while 26% of adenomas displayed scattered individual positive cells or a focal high concentration of positive cells. This later was more associated with severe dysplasia. Ras protein was detected in 37% of carcinomas and 32% of adenomas mostly throughout the tissue. p53 immunodetection was more frequent in adenomas originating in colons with synchronous carcinomas, particularly in patients with familial adenomatous polyposis and it may be a useful marker in these cases. Difference in the frequency of p53 and ras alterationbs was related to the location of the neoplasm. Immunodetection of p53 protein was correlated to the presence of a mutation in p53 gene at exon 7 and 5 in 4/6 carcinomas studied and 2 villous adenomas. Thus, we characterized in adenomas the abnormal expression of two proteins encoded by the most commonly altered genes in colorectal cancer. p53 alteration appears to be more specifically associated with transition to malignancy than ras. By using immunohistochemistry, a technique that keeps the architecture of the tissue intact, it was possible to correlate these alterations to histopathological characteristics that were associated with higher risks for transformation: villous content, dysplasia and size of adenoma. ^

Creation of a retinoblastoma mutant with dominant -negative activity

The Retinoblastoma tumor suppressor gene (RB) plays a role in a variety of human cancers. Experimental analyses have indicated that the protein product of the RB gene (pRb) plays a role in cell cycle regulation, and that this protein is required in cellular differentiation, senescence, and cell survival. pRb function is dependent on its ability to bind to cellular factors. There are multiple protein binding domains within pRb. Mutations within these domains which eliminate the ability of pRb to bind its targets result in loss of function. Loss of pRb function leads to tumorigenesis, although uncontrolled cellular proliferation is not a universal response to pRb inactivation. The ultimate response to the loss of pRb is influenced by both the genetic and epigenetic environments. Targeted disruption of RB in mice results in embryonic lethality, demonstrating the requirement for functional pRb in development. Close examination of various tissues from the embryos which lack wildtype RB shows problems in differentiation as well as showing induction of apoptosis. Although disruption of RB has provided useful information, complete inactivation of a gene precludes the possibility of discovering the functions that separate domains may have within the system. Creation of a dominant negative mutant by domain deletion whose phenotype is expressed in the presence of the wildtype may provide information about the intermediate functions of the protein. In addition, tissue specific targeting of a dominant negative mutant of pRb allows for comprehensive analysis of pRb function in organogenesis. In this thesis, a series of RB deletion mutants were created and tested for dominant negative activity as well as cellular localization. A tissue culture assay for dominant negative activity was developed which screens for the phenotype of apoptosis due to loss of pRb function. Two mutants from this series scored positive for dominant negative activity in this assay. The effect of these mutants within the assay environment can be explained by a model in which pRb acts as a facilitator of cell fate pathway decisions. ^

Alterations in the ras oncogene and the p53 tumor suppressor gene in ultraviolet radiation-induced skin carcinogenesis

A rapid increase of the ultraviolet radiation (UVR)-related skin cancer incidence has attracted more and more public attention during the last few decades. Prevention and treatment of UVR-related skin cancer has become an important public health issue in the United States. Recent studies indicate that mutations in ras and/or p53 genes may be involved in UVR-induced skin tumor development but the precise molecular mechanism remains unclear. In this study, alterations of H-ras and p53 genes were investigated in different stages of carcinogenesis in a chronic UVR (solar simulator) exposure-induced Sencar mouse skin carcinogenesis model in order to clarify the role of the alterations of these genes during the skin carcinogenesis process and to further understand the mechanisms by which UVR causes skin cancer.^ Positive ras-p21 staining in cell membranes and cytosol were detected in 18/33 (55%) of squamous cell carcinomas (SCCs), but were not detected in UV-exposed skin, papillomas, or spindle cell tumors (SCTs). Positive staining of the malignant progression marker K13 was found in 17/33 (52%) of SCCs only. A significant positive correlation was observed between the K13 and the ras-p21 expression. Polymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP) analysis and gene sequencing analysis revealed three point mutations, one (codon 56) in UV-exposed non-tumor bearing skin and the other two (codons 21 and 13) in SCCs. No UV-specific mutation patterns were found.^ Positive p53 nuclear staining was found in 10/37 (27%) of SCCs and 12/24 (50%) of SCTs, but was not detected in normal skin or papillomas. PCR-based SSCP and sequencing analysis revealed eight point mutations in exons 5 and 6 (four in SCTs, two in SCCs, and two in UV-exposed skin) including six C-T or C-A transitions. Four of the mutations occurred at a dipyrimidine (CC) sequence. The pattern of the mutations indicated that the mutagenic lesions were induced by UVR.^ These results indicate that overexpression of ras-p21 in conjunction with aberrant expression of K13 occurred frequently in UVR-induced SCCs in Sencar mouse skin. The point mutation in the H-ras gene appeared to be a rare event in UVR skin carcinogenesis and may not be responsible for overexpression of ras-p21. UVR-induced P53 gene alteration is a frequent event in UVR-induced SCCs and later stage SCT tumors in Sencar mice skin, suggesting the p53 gene mutation plays an important role in skin tumor malignant progression. ^

The p53 tumor suppressor pathway in culture and {\it in vivo\/}

p53 functions as a tumor suppressor through its ability to initiate either growth arrest or apoptosis in cells which have sustained DNA damage. p53 elicits these cellular phenotypes through its biochemical function as a transcriptional activator. By inducing the expression of a battery of target genes, p53 is able to prevent the propagation of cells with damaged DNA. However, the genes transcriptionally induced by p53 which have been identified to date do not fully explain p53 function. p53 has been demonstrated to activate genes involved in cell cycle inhibition, apoptosis and cell proliferation. The reasons for simultaneous activation of p53 targets with disparate, opposing functions are not clear, but may be due to the use of transformed cell lines in previous experiments. In the studies presented in this thesis, the pathway of p53 tumor suppression has been studied in detail in two systems chosen for their relevance to the natural cell environment. One utilizes a normal, unaltered cultured cell system; the other the whole mouse. In order to better understand the role of the known p53 targets in effecting p53 function in normal cells, early rat embryo fibroblasts were irradiated with ultraviolet light to induce DNA damage. It was discovered that p53 protein levels increased in response to irradiation. The known targets of p53, namely, $p21\sp{WAF1/CIP1},\ mdm2,\ cyclin\ G,$ and bax, were shown for the first time to have a differential temporal induction. The growth suppressor $p21\sp{WAF1/CIP1}$ was induced first, followed by cyclin G then mdm2, which is involved in proliferation through its inactivation of p53, and finally, the apoptosis promoter, bax. These findings indicated that p53 activates its target genes in a manner to allow maximum effectiveness of target function. The rat embryo fibroblasts were shown to undergo apoptosis 24 h after irradiation. Additionally, investigation of these cells for cell cycle alterations demonstrated a brief arrest in G1. In the second study, thymocytes from mice with wild type p53 were shown to undergo apoptosis and activate p53 target genes upon ionizing radiation treatment, while thymocytes from mice deficient in p53 could not. The p53 target genes mdm2 and fas were tested in vivo for their ability to mediate p53-regulated apoptosis, and were found dispensible for that cellular function. Therefore, the p53 targets identified to date do not fully explain the ability of p53 to function as a tumor suppressor. Potentially, functional redundancy between the known targets would account for the data seen in these experiments. Additionally, identification of additional target genes should add further understanding of the p53 pathway of tumor suppression. ^