A study of reduced penetrance retinoblastoma

Retinoblastoma is a pediatric tumor which is associated with somatic and inherited mutations at the retinoblastoma susceptibility locus, RB1. Although most cases of retinoblastoma fit the previously described 'two hit' model of oncogenesis, the molecular mechanisms underlying rare instances of familial retinoblastoma with reduced penetrance are not well understood. To better understand this phenomenon, a study was undertaken to uncover the molecular cause of low penetrance retinoblastoma in a limited number of families. In one case, a unique cryptic splicing alteration was discovered in the RB1 gene and demonstrated to reduce the level of normal RB1 mRNA produced. Penetrance in the large family known to carry this mutation is less than 50%. Data about the mutation supports a theory that reduced penetrance retinoblastoma is caused by partially functional mutations in RB1. In another family, three independent causes of retinoblastoma or the related phenotype of retinoma were indicated by linkage analysis, a finding unique in retinoblastoma research. A novel polymorphism restricted to Asian populations was also described during the course of this study. ^

The differential staurosporine mediated G1 arrest in normal versus tumor cells is dependent on the retinoblastoma and Chk1 proteins

The ability to regulate cell cycle progression is one of the differences that separates normal from tumor cells. A protein, which is frequently mutated or deleted in a majority of tumor cells, is the retinoblastoma protein (pRb). Previously, we reported that normal cells, which have a wild-type Rb pathway, can be reversibly arrested in the G1 phase of the cell cycle by staurosporine (ST), while tumor cells were unaffected by this treatment. As a result, ST may be used to protect normal cells against the toxic affects of chemotherapy. Here we set out to determine the mechanism(s) by which ST can mediate a reversible G1 arrest in pRb positive cells. To this end, we used an isogenic cell model system of normal human mammary epithelial cells (HMEC) with either intact pRb+ (p53-) or p53+ (pRb-) treated with ST. Our results show that pRb+ cells treated with low concentrations of ST, arrested in the G1 phase of the cell cycle; however, in pRb - cells there was no response. This was verified as a true G 1 arrest in pRb+ cells by two different methods for monitoring cell cycle kinetics and in two additional model systems for Rb (i.e. pRb -/- mouse embryo fibroblasts, and downregulation of RB with siRNA). Our results indicated that ST-mediated G1 arrest required pRb, which in turn initiated a cascade of events leading to inhibition of CDK4 and CDK2 activities and up-regulation of p21 protein. Further assessment of this pathway revealed the novel finding that Chk1 expression and activity were required for the Rb-dependent, ST-mediated G1 arrest. In fact, overexpression of Chk1 facilitated recovery from ST-mediated G1 arrest, an effect only observed in RB+ cells. Collectively, our data suggest pRb is able to cooperate with Chk1 to mediate a G1 arrest in pRb+ cells, but not in pRb- cells. The elucidation of this pathway can help identify novel agents that can be used to protect cancer patients against the debilitating affects of chemotherapy, by targeting only the normal proliferating cells in the body that are otherwise destroyed. ^

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. ^

Study of tumor suppressor genes p53 and Rb in acute myelogenous leukemia

Loss of antiproliferative function of p53 by point mutation occurred frequently in various solid tumors. However, the genetic change of p53 by deletion or point mutation was a rare event (6%) in the cells of 49 AML patients analyzed by single-stranded conformation polymorphism and sequencing. Despite infrequent point mutation, abundant levels of p53 protein were detected in 75% of AML patients studied by immunoprecipitation with p53 specific antibodies. Furthermore, p53 protein in most cases had an altered conformation as analyzed by the reactivity to PAb240 which recognizes mutant p53; p53 protein in mitogen stimulated normal lymphocytes also had similar altered conformation. This altered conformation may be another mechanism for inactivation of p53 function in the growth stimulated environment. Some evidence indicated that posttranslational modification by phosphorylation may contribute to the conformational change of p53.^ Retinoblastoma (Rb) gene inactivation by deletion, rearrangement or mutation has also been implicated in many types of solid tumors. Our studies showed that absence or low levels of Rb protein were observed in more than 20% of AML patients at diagnosis, and the low levels of Rb correlated with shorter survival of patients. The absence of Rb protein was due to gene inactivation in some cases and to abnormal regulation of Rb expression in others. ^

The effect of v-{\it mos\/} expression on the regulation of the {\it fos\/} promoter in 490N3T cells

The v-mos oncogene acquired by Moloney murine sarcoma viruses by recombination with the c-mos proto-oncogene encodes a 37kD cytoplasmic serine/threonine protein kinase which can phosphorylate tubulin and vimentin, as well as the cyclin B component of the maturation promotion factor complex (MPF). Our earliest experiments asked whether the v-mos protein could activate the transcription of transin. Since the transcription of transin was known to be mediated by both fos-dependent and fos-independent pathways, it seemed possible that the induction of transin transcription by v-mos might be mediated by p55$\sp{\rm c-}\sp{fos}$. Surprisingly, when we examined the effect of v-mos on the fos promoter, we observed a significant inhibition of transcription in 49ON3T cells, a subclone of N1H3T3 mouse fibroblasts.^ In this thesis we show that in mouse 49ON3T cells, transcription from the fos promoter is up to 10-fold repressed in the presence of v-mos. Moreover, in this cell line several other transforming constructs (v-ras, v-src, neu) also cause repression of the fos promoter. Interestingly, nontransforming oncogenes (e.g. myc) do not repress fos transcription. The repressive effect was lost in v-mos mutants lacking in ATP-binding or kinase domain, arguing that the effect on fos transcription was mediated by v-mos transforming kinase activity. As mos is a cytoplasmic protein, it was assumed that transcriptional repression was mediated by conversion of a transcriptional regulator to a repressor by mos-induced phosphorylation. As a first approximation of the identity of this factor, we mapped the position of the mos effect on the fos promoter using reporter (CAT) constructs. We found that repression was mediated by regions $-$221 to $-$106 and $-$122 to $-$65 relative to the fos transcriptional start site, both of which regions regulate baseline fos transcription. There are direct repeats containing E2F transcriptional activator/repressor recognition motifs in these regions which bind similar nuclear proteins independently of v-mos presence or absence. Our data show that the contribution of the direct repeat to baseline fos transcription is mediated by these E2F sites with perhaps some contribution from the overlapping retinoblastoma control element (RCE). We have shown that there is a separate DNA protein interaction in the direct repeat which is more pronounced in the presence of v-mos. The recognition site for this protein, which we speculate mediates the mos-induced downregulation of fos transcription, overlaps but is distinct from the E2F and RCE binding sites. (Abstract shortened by UMI.) ^

Mitotic mapping of a novel tumor suppressor locus on human chromosome 3q important in osteosarcoma tumorigenesis

Tumor-specific loss of constitutional heterozygosity by deletion, mitotic recombination or nondisjunction is a common mechanism for tumor suppressor allele inactivation. When loss of heterozygosity is the result of mitotic recombination, or a segmental deletion event, only a portion of the chromosome is lost. This information can be used to map the location of new tumor suppressor genes. In osteosarcoma, the highest frequencies of loss of heterozygosity have been reported for chromosomes 3q, 13q, 17p. On chromosomes 13q and 17p, allelic losses are associated with loss of function at the retinoblastoma susceptibility locus (RB1) and the p53 locus, respectively. Chromosome 3q is also of particular interest because the high percent of loss of heterozygosity (62%-75%) suggests the presence of another tumor suppressor important for osteosarcoma tumorigenesis. To localize this putative tumor suppressor gene, we used polymorphic markers on chromosome 3q to find the smallest common region of allele loss. This putative tumor suppressor was localized to a 700 kb region on chromosome 3q26.2 between the polymorphic loci D3S1282 and D3S1246. ^

Identification of the signal pathways modulated by expression of p210$\rm\sp{bcr-abl}$ and the inhibition of p210$\rm\sp{bcr-abl}$ transforming activity by polypeptides interacting with p210$\rm\sp{bcr-abl}$ in murine myeloid 32D cells

The Bcr-Abl fusion oncogene which resulted from a balanced reciprocal translocation between chromosome 9 and 22, t(9;22)(q11, q34), encodes a 210 KD elevated tyrosine specific protein kinase that is found in more than 95 percent of chronic myelogenous leukemia patients (CML). Increase of level of phosphorylation of tyrosine is observed on cell cycle regulatory proteins in cells overexpressing the Bcr-Abl oncogene, which activates multiple signaling pathways. In addition, distinct signals are required for transforming susceptible fibroblast and hematopoietic cells, and the minimal signals essential for transforming hematopoietic cells are yet to be defined. In the present study, we first established a tetracycline repressible p210$\rm\sp{bcr-abl}$ expression system in a murine myeloid cell line 32D c13, which depends on IL3 to grow in the presence of tetracycline and proliferate independent of IL3 in the absence of tetracycline. Interestingly, one of these sublines does not form tumors in athymic nude mice suggesting that these cells may not be completely transformed. These cells also exhibit a dose-dependent growth and expression of p210$\rm\sp{bcr-abl}$ at varying concentrations of tetracycline in the culture. However, p210$\rm\sp{bcr-abl}$ rescues IL3 deprivation induced apoptosis in a non-dose dependent fashion. DNA genotoxic damage induced by gamma-irradiation activates c-Abl tyrosine kinase, the cellular homologue of p210$\rm\sp{bcr-abl},$ and leads to activation of p38 MAP kinase in the cells. However, in the presence of p210$\rm\sp{bcr-abl}$ the irradiation failed to activate the p38 MAP kinase as examined by an antibody against phosphorylated p38 MAP kinase. Similarly, an altered tyrosine phosphorylation of the JAK1-STAT1 pathways was identified in cells constitutively overexpressing p210$\rm\sp{bcr-abl}.$ This may provided a molecular mechanism for altered therapeutic response of CML patients to IFN-$\alpha.$^ Bcr-Abl oncoprotein has multiple functional domains which have been identified by the work of others. The Bcr tetramerization domain, which may function to stabilize the association of the Bcr-Abl with actin filaments in p210$\rm\sp{bcr-abl}$ susceptible cells, are essential for transforming both fibroblast and hematopoietic cells. We designed a transcription unit encoding first 160 amino acids polypeptide of Bcr protein to test if this polypeptide can inhibit the transforming activity of the p210$\rm\sp{bcr-abl}$ oncoprotein in the 32D c13 cells. When this vector was transfected transiently along with the p210$\rm\sp{bcr-abl}$ expression vector, it can block the transforming activity of p210$\rm\sp{bcr-abl}.$ On the other hand, the retinoblastoma tumor suppressor protein (Rb), a naturally occurring negative regulator of the c-Abl kinase, the cellular homologue of Bcr-Abl oncoprotein, binds to and inhibits the c-Abl kinase in a cell cycle dependent manner. A polypeptide obtained from the carboxyl terminal end of the retinoblastoma tumor suppressor protein, in which the nuclear localization signal was mutated, was used to inhibit the kinase activity of the p210$\rm\sp{bcr-abl}$ in the cytoplasm. This polypeptide, called Rb MC-box, and its wild type form, Rb C-box, when overexpressed in the 32D cells are mainly localized in the cytoplasm. Cotransfection of a plasmid transcription unit coding for this polypeptide and the gene for the p210$\rm\sp{bcr-abl}$ resulted in reduced plating efficiency of p210$\rm\sp{bcr-abl}$ transfected IL3 independent 32D cells. Together, these results may lead to a molecular approach to therapy of CML and an in vitro assay system to identify new targets to which an inhibitory polypeptide transcription unit may be directed. ^

The utility of N5 for gene therapy of human cancer

Cancer is a result of defects in the coordination of cell proliferation and programmed cell death. The extent of cell death is physiologically controlled by the activation of a programmed suicide pathway that results in a morphologically recognizable form of death termed apoptosis. Inducing apoptosis in tumor cells by gene therapy provides a potentially effective means to treat human cancers. The p84N5 is a novel nuclear death domain containing protein that has been shown to bind an amino terminal domain of retinoblastoma tumor suppressor gene product (pRb). Expression of N5 can induce apoptosis that is dependent upon its intact death domain and is inhibited by pRb. In many human cancer cells the functions of pRb are either lost through gene mutation or inactivated by different mechanisms. N5 based gene therapy may induce cell death preferentially in tumor cells relative to normal cells. We have demonstrated that N5 gene therapy is less toxic to normal cells than to tumor cells. To test the possibility that N5 could be used in gene therapy of cancer, we have generated a recombinant adenovirus engineered to express N5 and test the effects of viral infection on growth and tumorigenicity of human cancer cells. Adenovirus N5 infection significantly reduced the proliferation and tumorigenicity of breast, ovarian, and osteosarcoma tumor cell lines. Reduced proliferation and tumorigenicity were mediated by an induction of apoptosis as indicated by DNA fragmentation in infected cells. We also test the potential utility of N5 for gene therapy of pancreatic carcinoma that typically respond poorly to conventional treatment. Adenoviral mediated N5 gene transfer inhibits the growth of pancreatic cancer cell lines in vitro. N5 gene transfer also reduces the growth and metastasis of human pancreatic adenocarcinoma in subcutaneous and orthotopic mouse model. Interestingly, the pancreatic adenocarcinoma cells are more sensitive to N5 than they are to p53, suggesting that N5 gene therapy may be effective in tumors resistant to p53. We also test the possibilities of the use of N5 and p53 together on the inhibition of pancreatic cancer cell growth in vitro and vivo. Simultaneous use of N5 and RbΔCDK has been found to exert a greater extent on the inhibition of pancreatic cancer cell growth in vitro and in vivo. ^

The role of deregulated E2F1 expression in brain tumors

Mutations disabling the retinoblastoma (Rb) pathway are among the most common in human cancers, including brain cancer. These mutations promote tumor development through deregulated control of the E2F family of transcription factors. E2F1 belongs to a class of E2F's identified as transcriptional activators and involved in the G1/S phase transition of the cell. However, E2F-1 presents with a paradox as it is considered to have membership in two gene classes, functioning as both an oncogene and a tumor suppressor. This unusual trait generates a degree of uncertainty on the role that E2F1 plays in the development or maintenance of any given tumor. Here we show that E2F1 functions as an oncogene in brain tumors through the generation of mice engineered to overexpress E2F1 specifically within glial cells and neuronal progenitors as directed by the GFAP promoter. Mice carrying the transgene develop with high penetrance a phenotype characterized by neurological deficits including paresia, ataxia, head tilt and seizures. MRI imagining of the tgE2F1 mice reveals a low incidence of mild hydrocephalus, and most notably, histological analysis demonstrates that 25% of tgE2F1 mice present with the spontaneous formation of malignant brain tumors. Overall these neoplasms show histological features from a wide range of aggressive brain cancers including medulloblastoma, choroid plexus carcinoma, primary neuroectodermic tumor and malignant gliomas. Isolation and characterization of astrocytes from the tgE2F1 animal reveals a highly proliferative population of cells with 55% ± 2.5 of the tgE2F1astrocytes, 35% ± 3.4 normal mouse astrocytes in S-phase and the acquired capacity to grow in anchorage independent conditions. Additionally tgE2F1 astrocytes show an aberrant phenotype with random chromosomal fusions and nearly all cells demonstrating polyploidy. Taken together, this model forces a comparison to human brain tumor formation. Mouse age as related to tumoral mimics the human scenario with juvenile tgE2F1 mice presenting embryonal tumors typically identified in children, and older tgE2F1 mice demonstrating gliomas. In this regard, this study suggests a global role for E2F1 in the formation and maintenance of multilineage brain tumors, irrefutably establishing E2F1 as an oncogene in the brain. ^

Repression of {\it neu} oncogene by E1A: Mechanisms and biological functions

The potential effects of the E1A gene products on the promoter activities of neu were investigated. Transcription of the neu oncogene was found to be strongly repressed by the E1A gene products and this requires that conserved region 2 of the E1A proteins. The target for E1A repression was localized within a 140 base pair (bp) DNA fragment in the upstream region of the neu promoter. To further study if this transcriptional repression of neu by E1A can inhibit the transforming ability of the neu transformed cells, the E1A gene was introduced into the neu oncogene transformed B104-1-1 cells and developed B-E1A cell lines that express E1A proteins. These B-E1A stable transfectants have reduced transforming activity compared to the parental B104-1-1 cell line and we conclude that E1A can suppress the transformed phenotypes of the neu oncogene transformed cells via transcriptional repression of neu.^ To study the effects of E1A on metastasis, we first introduced the mutation-activated rat neu oncogene into 3T3 cells and showed that both the neu oncogene transformed NIH3T3 cells and Swiss Webster 3T3 cells exhibited metastatic properties in vitro and in vivo, while their parental 3T3 cells did not. Additionally, the neu-specific monoclonal antibody 7.16.4, which can down regulate neu-encoded p185 protein, effectively reduced the metastatic properties induced by neu. To investigate if E1A can reduce the metastatic potential of neu-transformed cells, we also compared the metastatic properties of B-E1A cell lines and B104-1-1 cell. B-E1A cell lines showed reduced invasiveness and lung colonization than the parental neu transformed B104-1-1 cells. We conclude that E1A gene products also have inhibitory effect on the metastatic phenotypes of the neu oncogene transformed cells.^ The product of human retinoblastoma (RB) susceptibility gene has been shown to complex with E1A gene products and is speculated to regulate gene expression. We therefore investigated in E1A-RB interaction might be involved in the regulation of neu oncogene expression. We found that the RB gene product can decrease the E1A-mediated repression of neu oncogene and the E1A binding region of the RB protein is required for the derepression function. ^

Regulation of {\it neu\/} gene expression by the simian virus 40 large T antigen and tumor suppressors Rb and p53

The neu gene (also c-erbB-2 or HER2) encodes a 185 kilodalton protein that is frequently overexpressed in breast, ovarian and non-small cell lung cancers. Study of the regulation of neu indicates that neu gene expression can be modulated by c-myc or by the adenovirus 5 E1a gene product. This study demonstrates that the transforming protein, large T antigen, of the simian virus 40 represses neu promoter activity. Repression of neu by large T antigen is mediated through the region $-$172 to $-$79 (relative to first ATG) of the neu promoter--unlike through $-$312 to $-$172 for c-myc or E1a. This suggests a different pathway for repression of neu by large T antigen. The 10 amino acid region of large T required for binding the tumor suppressor, retinoblastoma gene product, Rb, is not necessary for repression of neu. Moreover, the tumor suppressors, Rb and p53 can independently inhibit neu promoter activity. Rb inhibits neu through a 10 base pair G-rich enhancer (GTG element) ($-$243 to $-$234) and also through regions close to transcription initiation sites ($-$172 to $-$79). Mutant Rb unable to complex large T is able to repress the region close to transcription initiation but not the GTG enhancer. Thus, Rb inhibits the two regulatory domains of the neu gene by different mechanisms. Both Rb and p53 can repress the transforming activity of activated neu in focus forming assays. These data provide evidence that tumor suppressors regulate expression of growth stimulatory genes such as neu. Therefore, one reason for the overexpression of neu that is frequently seen in breast cancer cells may be due to functional inactivation of Rb and p53 which is also a common occurrence in breast cancer cells. ^

Regulation of v-Mos kinase activity by phosphorylation

In this thesis, I investigated the effect of cylic AMP-dependent protein kinase (PKA) on v-Mos kinase activity. Increase in PKA activity in vivo brought about either by forskolin treatment or by overexpression of the PKA catalytic subunit resulted in a significant inhibition of v-Mos kinase activity. The purified PKA catalytic subunit was able to phosphorylate recombinant p37$\rm\sp{v-mos}$ in vitro, suggesting that the mechanism of in vivo inhibition of v-Mos kinase involves direct phosphorylation by PKA. Ser-263 was identified as a residue that is normally phosphorylated at a very low level but whose phosphorylation is dramatically increased upon forskolin treatment. Consistent with the inhibitory role of Ser-263 phosphorylation, the Ala-263 mutant of v-Mos was not inhibited by forskolin treatment. Based on our results, we propose that the known inhibitory role of PKA in the initiation of oocyte maturation could be explained at least in part by its inhibition of Mos kinase.^ Combining tryptic phosphopeptide two-dimensional mapping analysis and in vitro mutagenesis studies, I identified Ser-56 as the major in vivo phosphorylation site on v-Mos. I studied the interrelationship between Ser-34 and Ser-56 phosphorylation in regulating v-Mos function. After site-directed mutagenesis to substitute serine residues with alanine or glutamic acid in different combinations to mimick unphosphorylated and phosphorylated serines respectively, various v-Mos mutants were expressed in COS-1 cells. As expected, Ala-34 mutant of v-Mos had very low (less 5% of wild type) kinase activity. The Ala-56 mutant had kinase activity 50% that of wild type. Surprisingly, the Ala-34 Ala-56 double mutant and the Ala-56 mutant exhibited identical kinase activity. On the other hand, Ala-34 Glu-56 double mutant had reduced kinase activity comparable to Ala-34 mutant. These results suggest that the phosphorylation at Ser-56 may serve to inhibit the activation of newly synthesized Mos protein. As predicted from Xenopus c-Mos studies, Glu-34 mutant of v-Mos was highly active (125% that of wild type). Interestingly, consistant with the model involving an inhibitory role of Ser-56 phosphorylation, the Glu-34 Glu-56 double mutant was totally inactive as a kinase. Moreover in my experiments, there was a perfect correlation between the level of v-Mos kinase activity of various mutants and their transforming activity. The latter is dependent upon MEK1 phosphorylation/ activation in v-mos transformed cells. Residues corresponding to both v-Mos Ser-34 and Ser-56 are evolutionarily conserved in c-Mos. Therefore, the cytostatic factor function of c-Mos may be regulated in the same manner as v-Mos kinase activity.^ It has been known that v-mos transforms cells by affecting G1 phase progression of the cell cycle. Here I showed that mos induces cyclin D1 expression in mos transformed NIH 3T3 cells and NRK 6m2 cells, and this induced level was found to be unaffected by serum starvation. Consequently, cyclin D1-Cdk4 and cyclin E-Cdk2 activities increase, and retinoblastoma protein is hyperphosphorylated. Based on studies from several laboratories, these findings suggest that increased amount of cyclin D1-Cdk4 complexes ties up the limited amount of cyclin E-Cdk2 inhibitors (e.g. p27), causing the activation of cyclin E-Cdk2. My results indicate that activation of key cell cycle regulators of G1 phase may be important for cellular transformation by mos. (Abstract shortened by UMI.) ^