Gene Product Rb Research Articles

Retinoblastoma protein expressed in human non-Hodgkin's lymphoma cells generates resistance against radiation-induced apoptosis

Inactivation of the retinoblastoma susceptibility (Rb) gene has been observed in various cancers. Nevertheless, in several cancer cases, including non-Hodgkin's lymphoma, previous investigations showed that the Rb gene product (pRb) was abundantly expressed with varying degree. Here we report the THS-SP1.1 cell line isolated from non-Hodgkin's lymphoma. The THS-SP1.1 cells abundantly expressed pRb and showed resistance against radiation-induced apoptosis. Culture with the antisense phosphorothioate oligonucleotide complementary to the Rb gene augmented the apoptosis of THS-SP1.1 cells after radiation, whereas the control oligonucleotides did not. These data showed that pRb abundantly expressed in the lymphoma cells inhibited radiation-induced apoptosis.

INTERACTION SCREEN OF SP1 AND RB BINDING PROTEINS REGULATING SP1-MEDIATED TRANSCRIPTION 304

In an attempt to identify protein-protein interactions with the Sp1 transcription factor, we have used a modified yeast `two-hybrid' system to clone cDNAs encoding proteins that interact with both the retinoblastoma-susceptibility gene product (RB) and the ubiquitous transcription factor Sp1. Using the two-hybrid screening assay we have isolated and sequenced novel cDNA species that likely encode for proteins that directly regulate or contribute to the regulation of Sp1 by the Rb gene product. Our previous examination of the cyclin homologue of the human uracil-DNA glycosylase (UDG2) reveal tight and cell cycle-specific regulation by the Rb gene product through Sp1 -binding elements of the human UDG2 gene. UDG2 is suspect in the supression of tumors based on its chromosomal localization and has biochemical activity that inhibits cyclinE/cdk2 kinase activity. The timing of Rb -mediated regulation of Sp1 coincide with phosphorylation of Rb by cdk 2 and cdc2 kinases. Interestingly, some of the cDNAs we have cloned may represent proteins important for regulating the phosphorylation of both Sp1 and Rb. We believe this mechanism, contribute to the diverse and pleotropic actions rendered by Rb which may be used ultimately to control cell growth in response to cell growth and/or DNA damage through transcription mediated by Sp1.

Adding an Rb-binding site to an N-terminally truncated simian virus 40 T antigen restores growth to high cell density, and the T common region in trans provides anchorage-independent growth and rapid growth in low serum concentrations.

The simian virus 40 large T antigen is sufficient to confer on cells multiple transformed cell growth characteristics, including growth to a high cell density, rapid growth in medium containing low serum concentrations, and anchorage-independent growth. We showed previously that distinct regions of the protein were involved in conferring these properties and that removal of the first 127 amino acids of T antigen abrogated all three activities. At least three large-T-antigen transformation-related activities have been localized to that region: binding of the tumor suppressor gene product Rb and two independent activities contained within the common region shared by large T and small t antigens. The experiments described here were directed toward determining whether these were the only activities from the N terminus that were needed. To do so we reintroduced an Rb-binding region into the N-terminally truncated T antigen (T128-708) and examined the growth properties of cells immortalized by it in the presence and absence of small t antigen, which can provide the T-common-region transformation-related activities in trans. We show that an Rb-binding region consisting of amino acids 101 to 118, when introduced into a heterologous site in T128-708, is capable of physically binding Rb and that binding is sufficient for cells expressing the protein to acquire the ability to grow to a high saturation density. However, in low-serum medium, the growth rate of the cells and maximal cell density are reduced relative to those of wild-type-T-antigen-expressing cells, and the cells cannot divide without anchorage. This result suggests that although Rb binding is sufficient in the context of T128-708 to confer growth to a high density, one or more other N-terminally located T-antigen activities are needed for cells to acquire the additional growth properties. Small t antigen in trans supplied those activities. These results indicate that the T-common-region activities and Rb binding are the only activities from the T-antigen N terminus needed to restore full transforming activity to the N-terminally truncated T antigen.

Biphenotypic Blast Crisis of Chronic Myelogenous Leukemia: Abnormalities of p53 and Retinoblastoma Genes

The molecular mechanisms responsible for progression of chronic myelogenous leukemia (CML) to blast crisis have not been well defined. Blast crisis may be partially related to inac-tivation of tumor suppressor genes/such as p53 or retinoblastoma (Rb) gene. There is evidence for an association of blast cell phenotypes in CML with alterations of these genes: a strong association of myeloid phenotypes with abnormalities of the p53 gene and a weaker association of lymphoid phenotypes with abnormalities of the Rb system. We found a marked decrease in Rb gene product and rearrangements of the p53 gene simultaneously in two cases of biphenotypic blast crisis of CML (myeloid and B-lymphoid). These results support the association of blast cell phenotypes with alterations in tumor suppressor genes in CML blast crisis.

In VitroAntigene Therapy Targeting HPV-16 E6 and E7 in Cervical Carcinoma

Human papillomavirus (HPV) infection is believed to play a central role in cervical carcinogenesis. Specifically, two viral oncoproteins, E6 and E7, possess transforming ability and have been shown to interact with the cellular tumor suppressors p53 and p105, the retinoblastoma (Rb) gene product. To test the hypothesis that E6 and E7 play an active role in the maintenance of the malignant phenotype and may be ideal targets for antigene therapy, we tested the antiproliferative effects of phosphorothioate oligodeoxynucleotides (oligos) targeting HPV-16 E6 and E7 in cervical cancer cell lines and primary tumor explants.The ATP cell viability assay was used to measure growth effects of 27-mer antisense oligos targeting the ATG translational start region of HPV-16 E6 and E7 sequences in HPV-16-positive cell lines SiHa and CaSki and four advanced, primary cervical tumor explants. A random oligo sequence, an HPV-18-positive and HPV-negative cell line, one histologically confirmed endometrial and two ovarian tumors were used as negative controls. HPV type was confirmed by hybrid capture techniques. Cell lines and sterile (staging laparotomy) tumor cells were plated at 5000 cells/0.1 ml and 100,000 cells/0.5 ml in 96-well plates or soft agar, respectively, and incubated at 37°C with a single treatment of oligos at 0–16 μM. E6/E7 combinations at a fixed ratio of 1:1 were used at 0–8 μMfor each oligo. Cellular ATP was measured by luciferin/luciferase fluorescence on Day 6. HPV-16 E6 and E7 oligos showed antiproliferative effects in all HPV-16-positive cell lines and primary tumor explants (IC50s 6.9–9.5 μMfor cell lines, 9.1–12.1 μMprimary cervical tumors), while the HPV-negative C33-A cell line and HPV-18-positive cell line HeLa were relatively insensitive to the HPV-16 oligos (IC50s > 30 μMextrapolated). The endometrial and two ovarian primary tumors were also insensitive to the HPV E6 and E7 oligos (IC50s > 25 μMextrapolated). Random oligos had little effect on cell growth at concentrations up to 16 μM(< 25% inhibition), except in CaSki (@50% inhibition at 16 μM). Combinations of E6 and E7 demonstrated mixed synergistic and antagonistic effects as determined by combination indices (CI) derived from median effect parameters. In the HPV-16-positive primary cervical tumors and the cell line SiHa, E6/E7 combinations were synergistic at low doses (<25% growth inhibitory dose range) and antagonistic at doses above this. For the HPV-16-positive cell line CaSki, however, E6/E7 combinations were antagonistic at all dose ranges. Phosphorothioate oligos directed against the viral oncogenes E6 and E7 were shown to have antiproliferative effects specific to HPV-containing cancer cells. These specific antiproliferative effects suggest that HPV-16 E6 and E7 sequences play an active role in the malignant growth properties of cervical cancer cells and may be ideal targets for antigene therapy.

Prognostic Markers in Chondrosarcoma: Evaluation of Cell Proliferation and of Regulators of the Cell Cycle

Purpose. The prognosis, treatment principles and prediction of clinical outcome of patients with chondrosarcoma currently rest on histologic grading which is somewhat ambiguous due to difficulty in pathologic interpretation of this neoplasm. Immunohistochemistry, flow cytometry and oncogene/tumor suppressor gene expression have been examined as alternative indices to predict the biologic behavior of these tumors. Because of partial successes obtained with flow cytometry and because of the improvement in predicting recurrence offered by examining the S-phase fraction, we undertook the current study to determine if expression of specific regulators of the cell cycle would act as prognostic indicators for these patients. Subjects/methods. We examined archival pathologic specimens from 39 patients with at least 2 years' clinical follow-up for the presence of p53, Rb, src and MIB-1 by immunohistochemistry and correlated this with clinical histories and incidence of recurrence. Results. While Rb, p53 and src gene products were identified to a variable extent in these specimens, there was no prognostic significance to their expression. In contrast, MIB-1, an epitope expressed only during semiconservative replication and an accepted marker of cell proliferation, served as a significant prognostic indicator. MIB-1 staining was present in 14.5% of tumor cells in all specimens (range 0–59%). When MIB-1 staining was examined with respect to disease recurrence, there was a statistically significant association between staining and histologic grade (p < 0.05) as well as event-free survival (p < 0.02). Comparing survival curves stratified by MIB-1 expression, there was a significant decrease in event-free survival associated with increasing MIB-1 indices (p < 0.003). Covariates that were associated with event-free survival include histologic grade (p = 0.025) and stage (Musculoskeletal Tumor Society) (p = 0.014). There was no statistical association with patient age (p = 0.15), tumor size (p = 0.47), tumor histology (p = 0.62) or anatomic location (p = 0.316). Discussion. These results indicate that determination of the proliferation index by MIB-1 immunostaining may serve as a useful adjunct to current histopathologic classification. Patients with a high proliferation index may benefit from established adjuvant therapies or experimental approaches including immunotherapy or biologic modulation.

The CBP co-activator stimulates E2F1/DP1 activity.

The cell cycle-regulating transcription factors E2F1/DP1 activate genes whose products are required for S phase progression. During most of the G1 phase, E2F1/DP1 activity is repressed by the retinoblastoma gene product RB, which directly contacts the E2F1 activation domain and silences it. The E2F1 activation domain has sequence similarity to the N-terminal activation domain of E1A(12S), which contains binding sites for CBP as well as RB. Here, we present evidence that the CBP protein directly contacts E2F1/DP1 and stimulates its activation capacity. We show that CBP interacts with the activation domain of E2F1 both in vitro and in vivo. Deletion of four residues from the E2F1 activation domain reduces CBP binding as well as transcriptional activation, but still allows the binding of RB and MDM2. This deletion removes residues which are conserved in the N-terminal activation domain of E1A and which are required for the binding of CBP to E1A. When the E1A N-terminus is used as a competitor in squelshing experiments it abolishes CBP-induced activation of E2F1/DP1, whereas an E1A mutant lacking CBP binding ability fails to do so. These results indicate that CBP can act as a coactivator for E2F1 and suggest that CBP recognises a similar motif within the E1A and E2F1 activation domains. The convergence of the RB and CBP pathways on the regulation of E2F1 activity may explain the cooperativity displayed by these proteins in mediating the biological functions of E1A. We propose a model in which E1A activates E2F not only by removing the RB repression but also by providing the CBP co-activator.

Expression of oncogene products, anti-oncogene products and oncofetal antigens in intraductal papillary-mucinous neoplasm of the pancreas.

A few previous studies have demonstrated the expression or mutations of oncogenes and anti-oncogenes as well as that of oncofetal antigens in intraductal papillary-mucinous neoplasm of the pancreas. In this study, we have investigated the immunohistochemical expression of oncogene (ras and c-erbB-2) and anti-oncogene (p53 and retinoblastoma [Rb]) products and oncofetal antigens (CEA, CA19-9 and DUPAN-2) in nine such tumours of the pancreas. In normal pancreas (5 cases), the Rb gene product and CA19-9 were expressed in all cases, while ras and c-erbB-2 gene products, p53 protein, CEA and DUPAN-2 were not expressed. In intraductal papillary-mucinous tumours (n = 9), ras, c-erbB-2, p53 and Rb gene products were present in 4/9 (44%), 7/9 (78%), 0.9 (0%) and 6/9 (67%) cases, respectively. CEA, CA19-9 and DUPAN-2 were expressed in 8/9 (89%), 9/9 (100%) and 2/9 (22%) cases respectively. In invasive ductal adenocarcinoma of the pancrease (7 cases), ras, c-erbB-2, p53 and Rb gene products were expressed in 3/7 (43%), 6/7 (86%), 2/7 (29%) and 3/& (43%) cases respectively. CEA, CA19-9 and DUPAN-2 were expressed in 7/7 (100%), 7/7 (100%) and 6/7 (86%) cases, respectively. The extent and intensity of the expression of these antigens was greater in invasive ductal adenocarcinomas. These data suggest that activation of ras and c-erbB-2 oncogenes and inactivation of Rb anti-oncogene may contribute to the development and progression of intraductal papillary-mucinous tumours of the pancreas and that there is neo-expression of CEA and DUPAN-2 during the development and progression of these tumours.

Effect of interleukin-10 (IL-10) on IL-6 and PGE 2 production by murine decidual explants

Proliferation and differentiation of villous trophoblast during placental development, from an early stage to full-term, were investigated in routinely fixed and processed tissues, by means of the immunocytochemical localization of the cell cycle-related proto-oncogene c-myc and the p53 and retinoblastoma susceptibility (Rb) tumour-suppressor gene products. The proliferative activity of the trophoblast was determined using an antibody against proliferating cell nuclear antigen (PCNA) which stains all proliferating cells in paraffin-embedded tissues. Diffuse nuclear immunoreactivity for PCNA, c-myc and Rb gene products was a consistent finding in early cytotrophoblast; c-myc product expression was also detectable in both layers of mid-gestation trophoblast. Only scattered cytotrophoblastic nuclei of early gestational placenta displayed immunostaining for p53 gene product. In full-term placenta c-myc expression was undetectable while Rb gene product and PCNA immunoreactivity declined markedly. These results indicate that the expression of the above genes is spatio-temporally regulated during placental development. A potential involvement of the oncosuppressor gene products p53 and Rb in the control of trophoblastic proliferation and of c-myc in the control of both the proliferative and differentiation pathways of trophoblastic cells is suggested.

The E2F Transcription Factor Activates a Replication-Dependent Human H2A Gene in Early S Phase of the Cell Cycle

Histone gene expression is restricted to the S phase of the cell cycle. Control is mediated by a complex network of sequence-specific DNA-binding factors and protein-protein interactions in response to cell cycle progression. To further investigate the regulatory functions that are associated at the transcriptional level, we analyzed the regulation of a replication-dependent human H2A.1-H2B.2 gene pair. We found that transcription factor E2F binds specifically to an E2F recognition motif in the H2A.1 promoter region. Activation of the H2A.1 promoter by E2F-1 was shown by use of luciferase reporter constructs of the intergenic promoter region. Overexpression of the human retinoblastoma suppressor gene product RB suppressed E2F-1 mediated transcriptional activation, indicating an E2F-dependent regulation of promoter activity during the G1-to-S-phase transition. Furthermore, the activity of the H2A.1 promoter was also downregulated by overexpression of the RB-related p107, a protein that has been detected in S-phase-specific protein complexes of cyclin A, E2F, and cdk2. In synchronized HeLa cells, expression of luciferase activity was induced at the beginning of DNA synthesis and was dependent on the presence of an E2F-binding site in the H2A.1 promoter. Together with the finding that E2F-binding motifs are highly conserved in H2A promoters of other species, our results suggest that E2F plays an important role in the coordinate regulation of S-phase-specific histone gene expression.

The cytostatic function of c-Abl is controlled by multiple nuclear localization signals and requires the p53 and Rb tumor suppressor gene products.

c-Abl is a non-receptor protein-tyrosine kinase lacking a clear physiological role. A clue to its normal function is suggested by overexpression of Abl in fibroblasts, which leads to inhibition of cell growth. This effect requires tyrosine kinase activity and the Abl C-terminus. c-Abl is localized to the cell nucleus, where it can bind DNA, and interacts with the retinoblastoma protein, a potential mediator of the growth-inhibitory effect. Nuclear localization of Abl can be directed by a pentalysine nuclear localization signal in the Abl C-terminus. Here, we have identified two additional basic motifs in the Abl C-terminus, either of which can function independently of the pentalysine signal to localize Abl to the nucleus. Using a quantitative transfection assay, we show that both c-Abl and transforming Abl proteins inhibit entry into S phase and this effect is absolutely dependent on nuclear localization. Further, we demonstrate that the Abl cytostatic effect requires both the Rb and p53 tumor suppressor gene products. These results indicate that Abl inhibits cell proliferation by interacting with central elements of the cell cycle control apparatus in the nucleus, and suggest a direct connection between p53 and Rb in this growth-inhibitory pathway.

Interleukin-1 Induces Growth Arrest by Hypophosphorylation of the Retinoblastoma Susceptibility Gene Product RB

Interleukin-1 (IL-1) causes G0/G1 phase growth arrest in human melanoma cells, A375-C6. Because hypophosphorylation of the retinoblastoma susceptibility gene product, RB, is one of the key events responsible for G0/G1 phase growth arrest, we investigated whether IL-1 altered the phosphorylation status of RB protein in these cells. Exposure to IL-1 caused a time-dependent increase in hypophosphorylated RB that correlated with an accumulation of cells arrested in the G0/G1 phase. The ability of IL-1 to cause hypophosphorylation of RB and growth arrest was abrogated by the SV40 large T antigen, which binds preferentially to hypophosphorylated RB, but not by the K1 mutant of the T antigen, which is defective in binding to RB. Furthermore, the cells were protected from IL-1-inducible growth inhibition by ectopic expression of dominant-negative mutants of the Rb gene, or the transcription factor E2F-1, which is a downstream target of RB. These results suggest that hypophosphorylated RB mediates the growth arrest induced by IL-1.

Molecular cancer epidemiology--the present status and future possibilities

Molecular cancer epidemiology is a relatively new strategy for malignancies. This strategy has made it possible to diagnose the predisposition to cancer. An individual is said to have a predisposition to cancer when a tumor-suppressor gene is inactivated in germ-line cells. Mainly the inactivation of the tumor-suppressor gene is caused by mutations at the coding region of the gene. However, we clarified that point mutations or hypermethylations of the retinoblastoma tumor-suppressor gene (RB) also cause inactivation of the gene, resulting in retinoblastoma. On the other hand, studies to improve the diagnosed predisposition to cancer have not been performed. We therefore started a basic study for this purpose. As the first example of limiting the predisposition to cancer, the cases with a point mutation at the RB promoter region might be good candidates. In these carriers, only the RB promoter region is inactivated in spite of a lack of abnormalities in the coding region. Therefore, if the RB promoter activity is recovered by drugs, predisposition to retinoblastoma should be limited. As the second example, Li-Fraumeni syndrome in which the p53 gene is hereditarily mutated might be a good candidate. Recently p53 has been reported to stimulate the WAF1 gene, and the WAF1 protein to inhibit cdk activity, which inactivates the RB gene product by phosphorylation. In addition, we found that p53 up-regulates the promoter activity of the RB gene. These findings suggest that p53 directly or indirectly activates RB at the transcriptional or post-transcriptional level. Therefore, reactivation of the WAF1 or RB gene by certain drugs might compensate for the loss of function of p53 in Li-Fraumeni syndrome. We then suggest that it might be possible to prevent cancer by enhancing some intact target genes of the genetically inactivated tumor-suppressor gene. We term this new strategy "gene-regulating chemoprevention." To test this hypothesis it is important to clarify the structure of the RB promoter. In summary we found that RBF-1 and ATF sites are the core promoter regions, that the neighboring E2F site is a silencer site, and that E4TF1 preferentially binds to the RBF-1 site. We then speculate that drugs interfering with the binding of the E2F complex might become good candidates enhancing RB promoter activity. To find drugs regulating the promoters of these genes, it is reasonable to try G1 arresting drugs, because WAF1 and RB are thought to arrest cells at the G1 phase. Actually we found several drugs causing G1 arrest. They are several flavonoids which we ingest daily from vegetables and fruits, or prostaglandin D2 and its metabolite. In summary, we propose that "gene-regulating chemoprevention" will be a useful method for molecular cancer epidemiology.

Use of the E2F transcription factor by DNA tumor virus regulatory proteins.

In most cases the normal host cell for infection by the DNA tumor virus is a quiescent, terminally differentiated cell that is not dividing. Various experiments have demonstrated that upon infection these cells are stimulated to enter S phase, as indicated by the synthesis of cellular DNA and the induction of activities associated with DNA replication, particularly those enzymes involved in deoxynucleotide biosynthesis (Hatanaka and Dulbecco 1966; Ledinko 1968; Yamashita and Shimojo 1969; Dulbecco et al. 1965; Frearson et al. 1965, 1966; Hartwell et al. 1965; Kara and Weil 1967; Kit et al. 1966a, b, 1967a, b; Sheinin 1966). This viral-mediated S phase induction almost certainly reflects the need of these viruses to create an environment appropriate for viral DNA synthesis since the levels of deoxynucleotides are low in quiescent cells and normally rise only when cells are stimulated to enter S phase (Bjorklund et al. 1990; Engstrom et al. 1985). Thus, the normal host for infection by these viruses, a nongrowing cell, is not an environment conducive to DNA replication. The capacity of the DNA tumor viruses to drive a quiescent cell into S phase is dependent largely on the action of the viral regulatory proteins that include adenovirus E1A, SV40 T antigen, and human papillomavirus (HPV) E7. These are also viral proteins that possess oncogenic activity through their common ability to inactivate the retinoblastoma gene product Rb. Indeed, it is now clear that the ability of these viral proteins to promote entry into S phase, so as to create an environment that facilitates viral DNA replication, also results in a loss of cell growth control when a viral infection cannot proceed to completion. Recent developments have led to the realization that these viral proteins mediate these events through the activation of the E2F transcription factor, and studies of their interactions have provided considerable insight into the basic mechanisms of cell growth control and oncogenesis.

Regulation of retinoblastoma gene expression in hormone-dependent breast cancer.

Studies have shown an increased risk for breast cancer in the mothers of children suffering from retinoblastoma and osteosarcoma, suggesting a role for the retinoblastoma susceptibility (Rb) gene product in breast cancer. We now show that estradiol decreases the expression of Rb at the level of protein and messenger RNA (mRNA) in estrogen-dependent breast cancer cell lines. Treatment of MCF-7 cells with 10(-9) M estradiol for 48 h resulted in a 70% decrease in the level of Rb protein. Ribonuclease protection assays showed a 50% decrease in the steady state levels of Rb mRNA by 12 h and a 70% decrease in Rb mRNA by 24 h. Treatment with estradiol had no effect on the rate of Rb gene transcription or on Rb mRNA stability, but resulted in an increase in the steady state level of Rb mRNA in the nucleus. The effect of estradiol was inhibited by 10(-7) M 4-hydroxytamoxifen. In the absence of estradiol, the antiestrogens 4-hydroxytamoxifen and ICI 164,384 increased Rb mRNA by 50% over that in estrogen-depleted conditions. Estradiol regulation of Rb mRNA also occurred in other estrogen-dependent breast cancer cell lines. Insulin-like growth factor I, insulin, progestins, and epidermal growth factor had no effect on Rb expression. In summary, these results show that estradiol specifically regulates the expression of the Rb susceptibility gene product in hormone-dependent breast cancer by a posttranscriptional mechanism that occurs in the nucleus. The results from this study suggest that the negative regulation of Rb expression by estradiol, rather than Rb loss or mutation, may play an important role in breast carcinogenesis.

Suppression of Mutations in Two Saccharomyces cerevisiae Genes by the Adenovirus E1A Protein

The protein products of the adenoviral E1A gene are implicated in a variety of transcriptional and cell cycle events, involving interactions with several proteins present in human cells, including parts of the transcriptional machinery and negative regulators of cell division such as the Rb gene product and p107. To determine if there are functional homologs of E1A in Saccharomyces cerevisiae, we have developed a genetic screen for mutants that depend on E1A for growth. The screen is based on a colony color sectoring assay which allows the identification of mutants dependent on the maintenance and expression of an E1A-containing plasmid. Using this screen, we have isolated five mutants that depend on expression of the 12S or 13S cDNA of E1A for growth. A plasmid shuffle assay confirms that the plasmid-dependent phenotype is due to the presence of either the 12S or the 13S E1A cDNA and that both forms of E1A rescue growth of all mutants equally well. The five mutants fall into two classes that were named web1 and web2 (for "wants E1A badly"). Plasmid shuffle assays with mutant forms of E1A show that conserved region 1 (CR1) is required for rescue of the growth of the web1 and web2 E1A-dependent yeast mutants, while the N-terminal 22 amino acids are only partially required; conserved region 2 (CR2) and the C terminus are dispensable. The phenotypes of mutants in both the web1 and the web2 groups are due to a single gene defect, and the yeast genes that fully complement the mutant phenotypes of both groups were cloned. The WEB1 gene sequence encodes a 1,273-amino-acid protein that is identical to SEC31, a protein involved in the budding of transport vesicles from the endoplasmic reticulum. The WEB2 gene encodes a 1,522-amino-acid protein with homology to nucleic acid-dependent ATPases. Deletion of either WEB1 or WEB2 is lethal. Expression of E1A is not able to rescue the lethality of either the web1 or the web2 null allele, implying allele-specific mutations that lead to E1A dependence.

Murine models of neoplasia: functional analysis of the tumour suppressor genesRb-1 andp53

Loss of function of one or both of the two tumour suppressor genes p53 and RB-1 has been recognised as an important step in the development of a variety of human neoplasias for some time. By virtue of the ability to manipulate the genome of murine embryonic stem cells in culture, it has become possible to generate strains of mice which bear inactivations of the murine counterparts of these genes. This article attempts to bring together some of the many results obtained from these murine strains which are shedding light both on the normal role played by both of these genes and the consequences of their dysfunction. Surprisingly neither gene product is revealed to have an indispensable role at the level of the single cell. Hence, even though the Rb-1 gene product clearly has an important role in cell cycle regulation animals constitutively deficient in this gene develop relatively normally for the first 10 days of embryogenesis. It is only at and beyond this stage of development that a requirement for Rb-1 becomes clear, in the regulation of certain cell populations through control of both proliferation and apoptosis. That loss of function of Rb-1 is associated with tumorigenesis is confirmed by the development of tumours of the pituitary gland within heterozygotes. The retinas of these animals, the target organ for tumorigenesis in human RB-1 heterozygotes, remain unaffected. The majority of mice homozygous for an inactivating p53 mutation survive to birth, but then rapidly succumb to tumorigenesis. Heterozygotes also develop tumours, but with a delayed time course and altered spectrum. Analysis of several tissue types from the mutant animals has shown p53 to be crucial for the normal induction of apoptosis following DNA damage, and it is thought that failure of this process is a key predisposing step towards tumorigenesis within the mutant animals. Finally, studies on these and other transgenic strains have revealed interactions between pathways governed by these two genes. For example, the fate of Rb-1 deficient cells has been shown, in some tissues at least, to be dependent upon the functional status of p53.

Activity of RNA polymerase I transcription factor UBF blocked by Rb gene product.

The protein encoded by the retinoblastoma susceptibility gene (Rb) functions as a tumour suppressor and negative growth regulator. As actively growing cells require the ongoing synthesis of ribosomal RNA, we considered that Rb might interact with the ribosomal DNA transcription apparatus. Here we report that (1) there is an accumulation of Rb protein in the nucleoli of differentiated U937 cells which correlates with inhibition of rDNA transcription; (2) addition of Rb to an in vitro transcription system inhibits transcription by RNA polymerase I; (3) this inhibition requires a functional Rb pocket; and (4) Rb specifically inhibits the activity of the RNA polymerase I transcription factor UBF (upstream binding factor) in vitro. This last observation was confirmed by affinity chromatography and immunoprecipitation, which demonstrated an interaction between Rb and UBF. These results indicate that there is an additional mechanism by which Rb suppresses cell growth, namely that Rb directly represses transcription of the rRNA genes.

Bidirectional regulation of c-fos promoter by an oncogenic gip2 mutant of G alpha i2. A novel implication of retinoblastoma gene product.

G alpha i2 is a tissue-specific proto-oncogene product, whose activated mutant gip2 induces transformation through less defined downstream pathways. We found that c-fos promoter is a target of gip2 in multiple kinds of cells. Serum response element was shown to be the positive enhancer element that mediates gip2-induced c-fos expression. We further demonstrated that gip2 stimulates the negative silencer activity of the retinoblastoma (Rb) control element (RCE) and inhibits the c-fos promoter activity through RCE located in the c-fos promoter region. The effect of gip2 on RCE was shown to be mediated by the Rb gene product (pRb). Furthermore, gip2 augmented underphosphorylated active form of pRb by promoting pRb expression and by affecting the phosphorylation state of pRb. gip2 therefore propagates both positive and negative signals to the c-fos promoter through two different elements, and pRb mediates the negative signal of gip2. We conclude that gip2 has bifunctional roles in transformation which pRb critically regulates. Given that Rat-1 cells, which gip2 can transform, lack the sensitivity to the gip2/pRb-mediated negative pathway, this study provides a novel insight into oncogenesis by gip2 and its tissue specificity.

Role of Retinoblastoma Gene Product in p53-Mediated DNA Damage Response

The cellular response to DNA-damaging agents involves the activation of cell cycle checkpoints. Checkpoints provide a transient delay in cell cycle progression, presumably to allow time for the cell to repair the damage. A most important checkpoint, active in the G1 phase of the cell cycle, is mediated by the p53 tumor suppressor gene product. To investigate the role of downstream components of the cell cycle machinery in p53-mediated G1 arrest, the possible involvement of the RB gene product was examined. Rb and p53 proteins were studied by immunoprecipitation and Western blotting experiments in the presence and absence of DNA-damaging treatment. The phosphorylation status of Rb was altered following DNA damage in p53 wild-type cell lines, but was not altered in p53 mutant cell lines, nor in cell lines where p53 function was abrogated by viral gene products. These findings indicate that Rb probably plays a role in the activation of the p53-mediated checkpoint.