Antonio Giordano - US grants

The Adenovirus E1A oncoprotein has been shown to be an effective tool in the identification of protein factors that regulate fundamental cellular processes such as transcription, RNA processing, DNA replication and cell cycle regulation. Several cellular proteins implicated in growth regulation interact with the transforming region of E1A. These proteins have been found to have apparent molecular weights of 33kD, 60kD, 105kD, 107kD, 107kD, 130kD and 300kD. Four of the above mentioned proteins have now been identified. The 105kD protein (P105-Rb) is the product of the retinoblastoma susceptibility gene, the 107kD protein (p107) is structurally related to p105-Rb and the 60kD protein is the product of the cyclin A gene. In addition, several less abundant proteins appear to associate with E1A; one of these is the cyclin-dependent kinase 2 (cdk2), a 33kD protein that is closely related to the cell cycle regulating kinase cdc2. In this grant the focus will be on a gene recently cloned and characterized by us which encodes the E1A-associated protein, p130. We have demonstrated by sequence analysis that this protein is structurally related to the retinoblastoma protein and to p107. By utilizing the technology and reagents employed in our previous studies, we will undertake a systematic examination of the structure and function of p130, and assess the protein's role in regulating cellular proliferation and neoplastic transformation. The demonstration that p130 interacts with the transforming region of E1A and the fact that p130 is related to pRB suggests that this protein may play an important regulatory role in cell cycle progression. Further, p130, like p105-Rb, may prove to be target for inactivation by transforming viruses or by chromosomal aberrations which occur in a wide spectrum of neoplasm. The goals of this proposal are thus: (1) To prepare immunological reagents that will simplify the study of p130 and to identify new protein species that interact with p130. (2) To analyze p130 at different stages of the cell cycle. (3) To conduct structure/function studies of p130 (4) Genomic organization studies of p130.

Nearly 70% of the adult population is infected with the JC neurotropic polyoma virus. In immunocompromised individuals this results in the development of the fatal neurodegenerative disease, progressive multifocal leukoencephalopathy (PML). In several experimental animals, infection of brain with JCV results in the formation of glioblastoma. Intracerebral inoculation of golden Syrian hamsters with JCV preceded the development in a great (80%) proportion of these animals of malignant brain tumors or peripheral neuroblastomas. A similar phenotype of brain tumors was seen upon the inoculation of owl monkeys with JCV, with the development of malignant astrocytomas expressing the viral T-antigen. The ability of JCV to cause tumors in these animals is well documented. The JC virus is capable of transforming primary hamster brain cells and primary human fetal glial cells. The transforming ability of JCV is attributed to its multipotent early gene product, the large T-antigen. In vitro studies have indicated that the JCV T-antigen is capable of interaction with the tumor suppressor genes, p53, pRb, p107, and p130. T-antigen is thought to functionally inactivate these tumor suppressor genes in glial cells, resulting in the formation of glial tumors in these animals. In this proposal, we will use our unique JCV-injected newborn hamster system to study in vivo the molecular mechanisms involved in the formation and progression of brain tumors through the disruption of the key regulatory factors of the cell cycle. We will examine the role of the Rb family and how its interaction with JCV T-antigen results in the disruption of the regulatory processes of the cell cycle and allows for neoplastic transformation and tumorigenesis. In particular, we will focus on the pRb2/p130, the Rb family member first cloned in our laboratory and exhibits growth suppressive properties in glioma cells. Specifically we will: (1) Determine the biological importance of pRb2/p130 in the formation and progression of hamster gliomas; (2) Characterize the biochemical properties of pRb2/p130 associated with its interaction with JCV during cell growth and transformation, and the biochemical effects of this interaction on cyclins and cyclin-dependent kinases (cdks); and (3) structurally and functionally characterize the JCV T-antigen interaction with pRb2/p130 in an in vivo system.

Many forms of human malignancies have been linked to mutations in the tumor suppressor gene retinoblastoma, RB. The nuclear phosphoprotein, pRb, is involved indirectly in the cell cycle machinery by serving as a negative regulator in some specific cell types through its interactions with cellular proteins such as the transcription factor E2F. The importance of pRb in the inhibition of proliferation is evidenced by the necessity of a number of oncogenic human DNA viruses to encode functional oncoproteins, which can effectively bind and sequester pRb, in order to elicit a transformed phenotype in infected cells. The interaction of pRb with the viral oncoproteins as well as to E2F occurs at a specific "pocket region" in pRb. This pocket region is shared by two additional E1A associated proteins and has lead to the identification of the Rb-family of proteins, pRb, p107 and the newest member that we have recently cloned, pRb2/ p130. Recent functional studies of p107 and pRb2/pl30 have indicated that while the Rb-family members may be able to complement each other the proteins are not fully functionally redundant. Like pRb, ectopic expression of p107 is able to suppress the growth of certain cell lines. Additionally, pRb, p107, and p130/pRb2 form complexes with E2F. However, the temporal order of the complexes formation appears to vary. There is no evidence to support the notion that p107 is normally a tumor suppressor. Furthermore, the expression of p107 in cell lines derived from retinoblastoma tumors implies that p107 is unable to complement the lack of pRb and to suppress tumor formation. pRb2/p130, however, has been mapped to human chromosome 16q12.2, an area in which deletions have been found in several human neoplasias which is in support of an involvement of the RB2/p130 gene in human cancer as a tumor suppressor gene. The goals of this proposal are thus: (1) To assess the role of pRb2/p130 in cellular proliferation and tumorigenesis. (2) To analyze Rb2/p130 during growth arrest and apoptosis either coupled or uncoupled from the developmental program of terminal differentiation. (3) To investigate the interactions of Rb2/p130 with the IGF-1 pathway with regard to transcriptional and post-transcriptional modifications. (4) To investigate the interactions of pRb2/p130 with the Myb-family genes.

The retinoblastoma gene family currently consists of three members: pRb, p107, and pRb2/p130, that share a particular functional domain termed the "pocket" structure. The pocket region is responsible for many of the known specific functionality relevant protein-protein interactions in which these molecules are involved. All three family members have been shown to be growth suppressive nuclear phosphoproteins whose phosphorylation status is regulated in a cell cycle dependent manner. Ectopic expression of each of the family members leads to G1-growth arrest of sensitive cells. The importance of the Rb family in the inhibition of proliferation is evidence by the necessity of a number of oncogenic human DNA viruses to encode oncoproteins (E1A, T-antigen, and E7) which can effectively bind and sequester the Rb-family members to elicit a transformed phenotype in infected cells. The human Prb2/p130 gene maps to 16q12.2, a region found deleted in several human neoplasia. pRb2/p130 has been implicated in the pathogenesis and progression of several human cancers, including lung cancer, suggesting that the pRb2/p130 gene may be a tumor suppressor gene like RB. Despite their many similarities, however, it is becoming increasingly clear that even though the Rb family members may be able to complement each other, they are not fully functionally redundant. Each of the Rb family members associate with and modulate the function of distinct members of the E2F transcription factor family in a temporally modulated schedule. The T98G human glioblastoma cell line is refractory to the effects of pRb and p107 but undergoes growth arrest from pR2/p130, indicating the pRb2/p130 is not merely a surrogate for either pRb or p107 and that there are fundamental differences in the specific mechanisms of growth inhibition employed by the three family members. Additionally, unlike pRb, the phosphorylated form of pRb2/p130 is the preferred target of the DNA tumor viral oncoprotein E1A, suggesting that a different mechanism may underlie the functional regulation of pRb2/p130 and demonstrating that one can not use the Rb model to speculate the significance and regulation of phosphorylation of pRb2/p130. The goals of this proposal are thus: a. To define the growth inhibitory mechanism(s) employed by pRb2/p130, b. To understand the regulatory function of pRb2/p130 phosphorylation, c. To define the role of the Rb2/p130 gene in human lung cancer development and progression, d. To prepare viral models for studying pRB2/p130 in vivo.

DESCRIPTION (provided by applicant): HIV-1 has long been recognized as the etiological agent in acquired immunodeficiency syndrome (AIDS). Although neoplasms arise in HIV-1-infected patients more frequently than in other forms of immunosuppression, the role of HIV-1 as an oncogenic virus has not yet been clarified. HIV-1 encodes for the Tat transcription protein, which is essential for efficient viral replication. Tat is a likely candidate to contribute to tumor pathogenesis in HIV-1-infected patients because of its growth promoting activity, angiogenic function and regulation of the apoptotic pathway. The oncogenic role of Tat is further supported by an increased incidence of tumors in Tat-transgenic mice. 2-12 percent of AIDS patients develop primary central nervous system (CNS) lymphomas, of which 98 percent are B-cell lymphomas. Recently, a virus-linked mechanism of lymphomagenesis involving the Rb2/pl30 pathway has been proposed in AIDS-related Burkitt's lymphoma (BL). A deregulation of cell growth control by RB-related proteins may be the first step in lymphomagenesis in HIV-1-infected patients. However, little is known about the mechanisms by which HIV-1 gene products interact with the RB family and other cell cycle regulatory proteins. Among the latter, Cdk9 (PITALRE), identified and cloned in our laboratory, is the most likely candidate to be involved in the development of AIDS-related neoplasms. Cdk9, a cdc2-related kinase, promotes general elongation of transcription by activating the C-Terminal Domain (CTD) of RNA Polymerase II. Cdk9 is a partner of cyclin T1, which binds to the HIV Tat protein, supporting the positive involvement of Cdk9 in HIV replication and suggesting its possible role in the development of AIDS-related neoplasms. The goal of this proposal is to investigate the interaction between HIV gene products and cell cycle regulatory proteins and their role in the development/growth of the most common AIDS-related CNS and other subtype lymphomas. In particular, we will focus our attention on the cell cycle regulatory proteins, pRb2/pl30, Cdk9 and cycT1, which seem to be involved in AIDS-related tumorigenesis.

Project #2: Tumor Suppressor Rb Family/pRb2 in Medulloblastoma. The human neurotropic virus, JCV, is widespread in the human population and is the established etiologic agent of the fatal demyelinating disease of the central nervous system (CNS), Progressive Multifocal Leukoencephalopathy (PML). Like other papovaviruses, the JCV genome consists of circular double-stranded DNA that is separated into early and late coding sequences by the viral cell type-specific regulatory region. The viral early gene, T-antigen has important regulatory functions in orchestrating the viral lytic cycle and possesses the ability to interact with several important cellular proteins including the tumor suppressor protein, pRb pointing to the oncogenic potential of this virus. In support of this notion, earlier studies have revealed that JCV has the ability to induce neural origin tumors in several animal models. More recently, in collaboration with Dr. Khalili (Leader Project #1), a transgenic animal which developed tumor from external granular layer of cerebellun_ mimicking human medulloblastoma was created. The transgene which express T-antigen exhibited the ability to interact with pRb as well as p53 and that may de-regulate cell cycle pathway leading to evolution of tumor However, the underlying mechanism of transactivation of these two tumor suppressors, particularly pRb remains unknown. To better understand the molecular events involved in the genesis of T-antigen inducec medulloblastomas, we have developed cell lines from mouse tumors. Interestingly, similar to tumor tissue, not all cells expressed T-antigen, leading to speculation that as tumor cells develop, expression of T-antigen may no longer be needed and expression of T-antigen becomes silent. Loss of T-antigen expression which is concomitant with the lack of the hypophosphorylated form (active form) of pRb2/pl30 suggests that at the early stage of tumorigenesis, expression of T-antigen and its association with pRb2/pl30 functionally inactivates this protein, while at the latter stage of tumor development, the active form of pRb2/pl30 may be lost due to an increase in the level of phosphorylation of pRb2/pl30 and/or enhancement in its proteolytic degradation. The observation on murine medulloblastoma tumors is in accord with our earlier observation on human medulloblastoma that demonstrated detection of active pRb2/p 130 in JCV-associated medulloblastomas expressing T-antigen, but not in tumor cells lacking JCV and its early protein. As the biological function ot pRb2/pl30 is dictated at several stage, most notably through its partnership with p27 Kip1 and the E2F family, in this research proposal we will utilize the mouse model of medulloblastoma as well as a collection of well-characterized human medulloblastomas to decipher the molecular events involved in dysregulation o1 pRb2/pl30 during various stages of the cell cycle in vitro and during the course of tumor formation in the cerebellum of experimental animals. The outcome should provide useful information for therapeutic intervention.

DESCRIPTION (provided by applicant): pRb2/pl30: from the mechanisms to therapy. The retinoblastoma (RB) family proteins are negative cell-cycle regulators normally expressed in a number of adult tissues. Each RB protein binds to and modulates the activity of the E2F transcription factors that stimulate the transcription of genes needed to progress through the S phase. The RBs-E2Fs repressive complexes function in association with histone deacetylase (HDAC 1) which essentially repress transcription probably through deacetylation of histone tail that protrudes from nucleosome. The protein stability and the functional activity of each RB family protein can be hampered by gene silencing due to their promoter hypermethylation and/or by their post-translational modifications such as acetylation and phosphorylation. Although some indications are available about the role of epigenetic or post-translational control of pRb/pl05 functional activity, there are very few data about the other members of RB family. However, acetylation and phosphorylation may be not completely independent processes. Phosphorylation and acetylation status can influence also the protein stability by determining the probability of ubiquitin-mediated degradation, which it is though to be an early event in programmed cell death, possibly related to cell commitment to apoptosis. As a consequence, it has been shown that failure to the interior cleavage of pRb/p105 is associates with resistance to induction of apoptotic response. Up to now no similar data has been reported for pRb2/pl30. Deregulated cell proliferation together with suppressed apoptosis, constitute the minimal common platform upon which all neoplastic evolution occurs. The rationale for these studies is that the identification of significance of the epigenetic or post-translational modification on the function, stability, biochemical interactions and degradation of pRb2/pl30. We will pursue the following aims:/) Rb2/p130 negatively regulates the transcription of cell-cycle genes to exert its growth suppressive function. 2) Epigenetic events for pRb2/p130 gene silencing promoting tumor progression. 3) pRb2/pl30 post-translational modification during the induction of growth arrest and apoptotic response. 4) The cross talk between the pRb2/pl 30 growth suppressive and apoptotic function and the activated KRas oncogenic signals in the LSL-K-Ras G12D mouse model. These experiments will provide further insight into the post-translational modifications necessary to induce pRb2/p 130- dependent growth suppression and apoptosis. Finally, our results will be significant in that they will give us clues to the mechanisms underlying pRb2/p 130 activity and to characterize its role in tumorigenesis providing useful tools for design novel therapeutic strategies.