Retinoblastoma Tumor Suppressor Gene Research Articles

The Rb tumor suppressor is required for stress erythropoiesis.

The retinoblastoma tumor suppressor gene plays important roles in cell cycle control, differentiation and survival during development and is functionally inactivated in most human cancers. Early studies using gene targeting in mice suggested a critical role for pRb in erythropoiesis, while more recent experiments have suggested that many of the abnormal embryonic phenotypes in the Rb null mouse result from a defective placenta. To address this controversy and determine whether Rb has cell intrinsic functions in erythropoiesis, we examined the effects of Rb loss on red cell production following acute deletion of pRb in vitro and under different stress conditions in vivo. Under stress conditions, pRb was required to regulate erythroblast expansion and promote red cell enucleation. Acute deletion of Rb in vitro induced erythroid cell cycle and differentiation defects similar to those observed in vivo. These results demonstrate a cell intrinsic role for pRb in stress erythropoiesis and hematopoietic homeostasis that has relevance for human diseases.

Activation of protein kinase C promotes human cancer cell growth through downregulation of p18(INK4c).

p18(INK4c), a member of INK4 family of cyclin-dependent kinase inhibitors, negatively regulates the cyclin D-cyclin-dependent kinase 4/6 complexes which promote G1/S transition by phosphorylating the retinoblastoma tumor-suppressor gene product. Several recent studies using p18(INK4c)-null mice revealed that the p18(INK4c) plays an important role in cell proliferation and tumor development. We report here that 12-O-tetradecanoylphorbol-13-acetate (TPA), widely used as a protein kinase C (PKC) activator, suppresses the expression of p18(INK4c) through its promoter, accompanied by the induction of human cancer cell growth. Reduction of p18(INK4c) using small interfering RNA (siRNA) also enhanced cell growth, suggesting that p18(INK4c) is a critical target of TPA. Ro 31-8425, a potent and highly specific PKC inhibitor abrogated the suppressive effect of TPA on p18(INK4c) gene expression. However, the expression of dominant-negative c-Jun (TAM-67) did not inhibit the action of TPA on p18(INK4c). These findings suggest that activation of PKC promotes human cancer cell growth through downregulation of p18(INK4c) in an AP-1 activation-independent manner. These results suggest that the accelerated cellular proliferation of some human tumors caused by enhanced PKC activity at least partially involves the suppression of p18(INK4c), which is a ubiquitously expressed cyclin-dependent kinase inhibitor.

Rapid identification of germline mutations in retinoblastoma by protein truncation testing.

To demonstrate the utility of protein truncation testing (PTT) for rapid detection and sequencing of germline mutations in the retinoblastoma tumor suppressor gene (RB1). We performed PTT, a technique based on the in vitro synthesis of protein from amplified RNA, on 27 probands from 27 kindreds with hereditary retinoblastoma. In 4 kindreds, PTT was also performed on 1 additional affected relative. Ten unrelated patients without retinoblastoma were included as negative control subjects. All PTT-detected mutations were further analyzed by focused sequencing of genomic DNA. When no mutation was detected by PTT, we performed exon-by-exon sequencing, as well as cytogenetic analysis by Giemsa-trypsin-Giemsa banding and by fluorescent in situ hybridization for RB1. The results of proband testing were used for direct genetic testing by polymerase chain reaction and sequencing in 11 relatives from 7 of the 27 kindreds. Of the probands tested, 19 (70%) of 27 tested positive for germline mutations by PTT. In 1 kindred, the proband had negative PTT results but an additional affected relative had positive PTT results. Focused DNA sequencing of 1 patient with positive PTT results from each of the 20 kindreds with positive PTT results revealed truncating mutations in 19 kindreds. Four demonstrated frameshift deletions, 6 had splice site mutations, and 9 showed nonsense mutations. Further analysis by genomic exon-by-exon sequencing and karyotype analysis of the 8 probands who tested negative for germline mutations by PTT revealed 1 splice site mutation, 2 missense mutations, and 1 chromosomal deletion. Focused sequencing based on positive PTT results was successfully used to confirm shared truncating mutations in additional affected family members in 2 kindreds. Using a multitiered approach to genetic testing, 23 (85%) of 27 kindreds had mutations identified and those detected by PTT received a positive result in as few as 7 days. In control subjects, PTT produced no false-positive results. Protein truncation testing is an effective, rapid single-modality screen for germline mutations in patients with retinoblastoma. When used as an initial screen, PTT can increase the yield of additional testing modalities, such as sequencing and chromosomal analysis, providing a timely and cost-effective approach for the diagnosis of heritable germline mutations in patients with retinoblastoma.Clinical Relevance The clinical application of PTT in retinoblastoma will improve detection of germline retinoblastoma mutations, which will supply critical information for prognosis, treatment planning, follow-up care, and genetic counseling.

Methylation Status of the Retinoblastoma Gene (RB1) in Osteosarcoma: No Evidence for Hypermethylation

Alterations of the retinoblastoma (RB1) tumor suppressor gene are not only associated with retinoblastoma but also with several other malignancies including osteosarcoma. Besides direct sequence alterations, hypermethylation of a CpG island in the promoter region can cause inactivation of the RB1 gene as it has been shown in retinoblastomas. We examined the methylation status of the RB1 gene in 25 osteosarcoma specimens by using the methylation-sensitive restriction enzymes SacII and SmaI. The restriction fragments were hybridized with clone p123, which is a 1.8-kb genomic subclone that spans the RB1 CpG island including the promoter region and exon 1. Whereas we reconfirmed hypermethylation of the RB1 gene in a sporadic retinoblastoma, no hypermethylation could be detected in the 25 osteosarcoma specimens, suggesting that hypermethylation of the RB1 promoter is not of major importance during osteosarcoma genesis.

Retinoblastoma gene expression in human non-melanoma skin cancer.

The aberrant expression of both the retinoblastoma and p53 tumor suppressor genes has been associated with more aggressive tumors, metastasis and lower survival. We have evaluated immunohistochemically the expression of pRB in a panel of non-melanoma skin cancers containing p53 somatic mutations. Nuclear anti-p53 staining was detected in 18 (72%) differentiated squamous cell carcinomas, six (100%) undifferentiated squamous cell carcinomas and seven (28%) basal cell carcinomas. A correlation was observed between p53 expression and the proliferative activity of differentiated squamous cell carcinomas (P < 0.066), undifferentiated squamous cell carcinomas (P < 0.05) and basal cell carcinomas (P < 0.01). Tumors were selected for mutant p53 expression by PCR-directed DNA sequencing and pRB expression measured immunohistochemically. Anti-pRB reactivity was detected in the nuclei of basal and suprabasal layer cells of normal epidermis, and in the proliferative compartment of all the differentiated squamous cell carcinomas, and basal cell carcinomas. A correlation was observed between pRB expression and the proliferative activity of the differentiated squamous cell carcinomas (P < 0.01) and basal cell carcinomas (P < 0.025). However, anti-pRB reactivity was not detected in the six anti-p53 reactive undifferentiated squamous cell carcinomas.

Rb and N-ras function together to control differentiation in the mouse.

The product of the retinoblastoma tumor suppressor gene (Rb) can control cell proliferation and promote differentiation. Murine embryos nullizygous for Rb die midgestation with defects in cell cycle regulation, control of apoptosis, and terminal differentiation of several tissues, including skeletal muscle, nervous system, and lens. Previous cell culture-based experiments have suggested that the retinoblastoma protein (pRb) and Ras operate in a common pathway to control cellular differentiation. Here we have tested the hypothesis that the proto-oncogene N-ras participates in Rb-dependent regulation of differentiation by generating and characterizing murine embryos deficient in both N-ras and Rb. We show that deletion of N-ras rescues a unique subset of the developmental defects associated with nullizygosity of Rb, resulting in a significant extension of life span. Rb(-/-); N-ras(-/-) skeletal muscle has normal fiber density, myotube length and thickness, in contrast to Rb-deficient embryos. Additionally, Rb(-/-); N-ras(-/-) muscle shows a restoration in the expression of the late muscle-specific gene MCK, and this correlates with a significant potentiation of MyoD transcriptional activity in Rb(-/-); N-ras(-/-), compared to Rb(-/-) myoblasts in culture. The improved differentiation of skeletal muscle in Rb(-/-); N-ras(-/-) embryos occurs despite evidence of deregulated proliferation and apoptosis, as seen in Rb-deficient animals. Our findings suggest that the control of differentiation and proliferation by Rb are genetically separable.

How the other half lives, the amino-terminal domain of the retinoblastoma tumor suppressor protein.

The retinoblastoma tumor suppressor gene (RB1) is currently the only known gene whose mutation is necessary and sufficient for the development of a human cancer. Mutation or deregulation of RB1 is observed so frequently in other tumor types that compromising RB1 function may be a prerequisite for malignant transformation. Identifying the molecular mechanisms that provide the basis for RB1-mediated tumor suppression has become an important goal in the quest to understand and treat cancer. The lion's share of research on these mechanisms has focused on the carboxy-terminal half of the RB1 encoded protein (pRB). This focus is with good reason since this part of the protein, now called the "large pocket," is required for most of its known activities identified in vitro and in vivo. Large pocket mediated mechanisms alone, however, cannot account for all observed properties of pRB. The thesis presented here is that the relatively uncharacterized amino-terminal half of the protein makes important contributions to pRB-mediated tumor suppression. The goals of this review are to summarize evidence indicating that an amino-terminal structural domain is important for pRB function and to suggest a general hypothesis as to how this domain can be integrated with current models of pRB function.

Acute mutation of retinoblastoma gene function is sufficient for cell cycle re-entry.

Cancer cells arise from normal cells through the acquisition of a series of mutations in oncogenes and tumour suppressor genes. Mouse models of human cancer often rely on germline alterations that activate or inactivate genes of interest. One limitation of this approach is that germline mutations might have effects other than somatic mutations, owing to developmental compensation. To model sporadic cancers associated with inactivation of the retinoblastoma (RB) tumour suppressor gene in humans, we have produced a conditional allele of the mouse Rb gene. We show here that acute loss of Rb in primary quiescent cells is sufficient for cell cycle entry and has phenotypic consequences different from germline loss of Rb function. This difference is explained in part by functional compensation by the Rb-related gene p107. We also show that acute loss of Rb in senescent cells leads to reversal of the cellular senescence programme. Thus, the use of conditional knockout strategies might refine our understanding of gene function and help to model human cancer more accurately.

A maize histone deacetylase and retinoblastoma-related protein physically interact and cooperate in repressing gene transcription.

In mammalian cells the product of the human retinoblastoma tumour suppressor gene (pRb) can recruit Rpd3-like histone deacetylases to repress transcription. In this study, we investigated whether this mechanism might also be relevant in plants and found both conserved and distinct features. The expression profiles of the Zea mays Rpd3-type histone deacetylase (ZmRpd3I) and the retinoblastoma-related (ZmRBR1) homologues were analysed during endosperm development. GST pull-down and immunoprecipitation experiments showed a physical interaction between ZmRBRI and ZmRpd3I. Because ZmRpd3I lacks a LXCXE motif, conserved in several pRb-interacting proteins, we have mapped the amino acid domains involved in the ZmRBR1/ZmRpd3I interaction. Furthermore, we observed that ZmRbAp1, a maize member of the MSI/RbAp family, facilitated this protein interaction. Co-transformations of tobacco protoplasts with plasmids expressing ZmRBRI and ZmRpd3I showed that the two proteins cooperate in repressing gene transcription. Our findings represent the first indication that in plants a regulator of important biological processes, ZmRBRI, can recruit a histone deacetylase, ZmRpd3I, to control gene transcription.

ARF mutation accelerates pituitary tumor development in Rb+/- mice.

Mice heterozygous for the retinoblastoma (Rb) tumor suppressor gene develop pituitary and thyroid tumors with high penetrance. We demonstrate here that loss of the ARF tumor suppressor strongly accelerates intermediate lobe pituitary tumorigenesis in Rb heterozygous mice. These effects in the pituitary are greater than those conferred by p53 loss in that Rb+-;ARF-- mice display significantly more early atypical lesions than Rb+-; p53-- mice. Also, Rb+-;ARF-- compound mutants do not develop many of the novel tumors or precancerous lesions seen in Rb+-;p53-- compound mutants. Although complete loss of ARF expression is not obligatory for pituitary tumorigenesis in Rb+- mice, alterations of the ARF locus are observed in tumors from Rb+-;ARF+- mice, consistent with a selective advantage of ARF inactivation in this context. We conclude that inactivation of ARF acts more broadly than that of p53 in connecting abrogation of the Rb pathway to tumorigenesis.

P130/p107 expression distinguishes adipogenic potential in primary myoblasts based on age

Recent investigations have provided significant evidence that many mesodermally derived tissues contain stem cell-like precursors capable of being stimulated to undergo differentiation into a variety of cellular lineages. We have recently reported that primary myoblasts isolated from 23-month-old mice have an increased adipogenic potential when compared to their 8-month-old counterparts. To further characterize the degree of adipocyte differentiation in these myoblasts, we examined early and late markers of adipocyte differentiation. Within the first 24 h of adipocyte differentiation, expression of p130 and p107, two members of the retinoblastoma tumor suppressor gene family, are regulated and this event is an important one early in adipogenesis. Consistent with the increased adipogenic potential of the older myoblasts and in contrast to the younger cells, the p130:p107 pattern of expression is very similar to that observed in adipogenesis where there is a transient increase in p107 expression accompanied by a decrease in p130 expression. Interestingly, while these older cells accumulated lipid and expressed genes associated with lipid metabolism, they failed to express adipsin and leptin, two well-established markers of terminal adipocyte differentiation. These results suggest that older myoblasts are capable of initiating and progressing through the adipogenic program to a point where they express genes associated with lipid metabolism, but do not reach a terminally differentiated state. This finding may have important metabolic implications in the aging population.

Preimplantation-embryo-specific cell cycle regulation is attributed to the low expression level of retinoblastoma protein

It is known that a characteristic of the mammalian preimplantation-embryo-specific cell cycle is the substantially shortened G1-phase, although the regulation mechanisms of the unique cell cycle remain unclear. In the present study, we first examined the presence of retinoblastoma (RB) tumor suppressor gene product throughout mouse preimplantation embryo development and found that the RB expression was down-regulated between the four-cell and morula stages. Furthermore, the overexpression of RB protein in the mouse embryos during this phase inhibited their development significantly. These results suggest that the absence of RB protein contributes to the preimplantation-embryo-specific cell cycle.

Molecular cloning and characterization of the human p19 INK4d gene promoter

p19 INK4d, a member of the INK4 family of cyclin-dependent kinase (CDK) inhibitors, negatively regulates the cyclin D–CDK4/6 complexes, which promote G1/S transition by phosphorylating the retinoblastoma tumor-suppressor gene product. To investigate the mechanism of transcriptional regulation of the p19 INK4d gene, we characterized the 5′-flanking region of the human p19 INK4d gene. The cap-site hunting method revealed that the transcription starts at −16 nucleotide (nt) upstream of the initiation codon. The 5′-flanking region of the human p19 INK4d gene was ligated to a luciferase reporter gene and possessed functional promoter activity. Luciferase assay with a series of truncated 5′-flanking regions indicated that the region from −81 to −2 nt could drive the transcription of the p19 INK4d gene. Several Sp1 and activating protein 2 binding sites are located within the region from −81 to −2 nt. Mutation of the second Sp1 binding site from −33 to −25 nt decreased the promoter activity. Collectively, it was demonstrated that the human p19 INK4d gene is under the control of TATA-less promoter and the Sp1 binding site is involved in the transcription.

The Use Of Real-Time Quantitative Polymerase Chain Reaction To Detect Hypermethylation Of The Cpg Islands In The Promoter Region Flanking The Gstp1 Gene To Diagnose Prostate Carcinoma

We developed a real-time, quantitative, methylation sensitive polymerase chain reaction (PCR) protocol to analyze hypermethylation of the CpG islands in the promoter region of the pi class glutathione-S-transferase gene GSTP1 in prostate cancer tissue. A total of 21 prostate cancer and 72 benign prostate hyperplasia (BPH) tissue samples were analyzed. Genomic DNA was digested with restriction enzyme, followed by real-time quantitative PCR amplification. Cycle threshold values were used to determine whether cancer genome was present in these tissues. A cutoff cycle threshold value of 35 was arbitrarily assigned. Samples with a cycle threshold of 35 or less were considered positive for prostate cancer. Conventional nested PCR was also performed for comparison. The mean cycle threshold values plus or minus standard deviation in prostate cancer and BPH cases were 30.12 +/- 2.88 and 37.77 +/- 2.72, respectively. All prostate cancer samples analyzed showed positive results, while 5 of the 72 BPH samples tested positive. Conventional nested PCR data indicated that 19 of 21 prostate cancer cases were positive for the methylation change, while 71 of 72 BPH cases tested negative. The test limitations of real-time PCR and the nested PCR protocols were determined to be 0.048 and 0.64 ng. DNA, respectively. We established a novel protocol for detecting the methylation change in the 5' regulatory sequence flanking the GSTP1 gene. The sensitivity of this protocol was superior to that of conventional nested PCR. The data also suggest that this novel protocol may accurately discriminate prostate carcinoma from BPH.

CD28 costimulation mediates down-regulation of p27kip1 and cell cycle progression by activation of the PI3K/PKB signaling pathway in primary human T cells.

CD28 provides a costimulatory signal that cooperates with the TCR/CD3 complex to induce T cell activation, cytokine production, and clonal expansion. We have recently shown that CD28 directly regulates progression of T lymphocytes through the cell cycle. Although a number of signaling pathways have been linked to the TCR/CD3 and to CD28, it is not known how these two receptors cooperate to induce cell cycle progression. Here, using cell-permeable pharmacologic inhibitors of phosphatidylinositol 3-hydroxykinase (PI3K) and mitogen-activated protein kinase kinase (MEK1/2), we show that cell cycle progression of primary T lymphocytes requires simultaneous activation of PI3K- and MEK1/2-dependent pathways. Decreased abundance of cyclin-dependent kinase inhibitor p27(kip1), which requires simultaneous TCR/CD3 and CD28 ligation, was dependent upon both MEK and PI3K activity. Ligation of TCR/CD3, but not CD28 alone, resulted in activation of MEK targets extracellular signal-related kinase 1/2, whereas ligation of CD28 alone was sufficient for activation of PI3K target protein kinase B (PKB; c-Akt). CD28 ligation alone was also sufficient to mediate inactivating phosphorylation of PKB target glycogen synthase kinase-3 (GSK-3). Moreover, direct inactivation of GSK-3 by LiCl in the presence of anti-CD3, but not in the presence of anti-CD28, resulted in down-regulation of p27(kip1), hyperphosphorylation of retinoblastoma tumor suppressor gene product, and cellular proliferation. Thus, inactivation of the PI3K-PKB target GSK-3 could substitute for CD28 but not for CD3 signals. These results show that the PI3K-PKB pathway links CD28 to cell cycle progression and suggest that p27(kip1) integrates mitogenic MEK- and PI3K-dependent signals from TCR and CD28 in primary T lymphocytes.

Single nucleotide polymorphism detection by combinatorial fluorescence energy transfer tags and biotinylated dideoxynucleotides.

Combinatorial fluorescence energy transfer (CFET) tags, constructed by exploiting energy transfer and combinatorial synthesis, allow multiple biological targets to be analyzed simultaneously. We here describe a multiplex single nucleotide polymorphism (SNP) assay based on single base extension (SBE) using CFET tags and biotinylated dideoxynucleotides (biotin-ddNTPs). A library of CFET-labeled oligonucleotide primers was mixed with biotin-ddNTPs, DNA polymerase and the DNA templates containing the SNPs in a single tube. The nucleotide at the 3'-end of each CFET-labeled oligonucleotide primer was complementary to a particular SNP in the template. Only the CFET-labeled primer that is fully complementary to the DNA template was extended by DNA polymerase with a biotin-ddNTP. We isolated the DNA extension fragments that carry a biotin at the 3'-end by capture with streptavidin-coated magnetic beads, while the unextended primers were eliminated. The biotinylated fluorescent DNA fragments were subsequently analyzed in a multicolor fluorescence electrophoresis system. The distinct fluorescence signature and electrophoretic mobility of each DNA extension product in the electropherogram coded the SNPs without the use of a sizing standard. We simultaneously distinguished six nucleotide variations in synthetic DNA templates and a PCR product from the retinoblastoma tumor suppressor gene. The use of CFET-labeled primers and biotin-ddNTPs coupled with the specificity of DNA polymerase in SBE offered a multiplex method for detecting SNPs.

Cyclin D1 amplification and p16(MTS1/CDK4I) deletion correlate with poor prognosis in head and neck tumors.

Cyclin D1, a cell cycle regulator localized to chromosome 11q13, is amplified in several human tumors including head and neck squamous cell carcinoma (HNSCC). Amplification and/or overexpression of cyclin D1 have been correlated to a poor prognosis. Deletion of the p16 gene, localized to 9p21, has also been observed in a significant proportion of HNSCC. The p16 gene regulates cyclin D1-CDK4 activity and prevents retinoblastoma tumor suppressor gene phosphorylation, thereby downregulating cellular proliferation. Detection of cyclin D1 amplification and p16 deletion using a simple and sensitive method will be valuable for the development of effective treatment modalities for head and neck cancer. We have used fluorescence in situ hybridization (FISH) to study cyclin D1 amplification and p16 gene deletion in head and neck tumors. Both single- and dual-color FISH were performed. Paraffin-embedded tissues from 103 patients with HNSCC were analyzed using genomic DNA probes for cyclin D1 and p16. Dual-color FISH was performed with chromosome 11 or 9 centromeric probes as a control. Twenty-eight of these samples were analyzed for p16 expression by immunohistochemistry. Cyclin D1 amplification was observed in 30% (31/103) of patients, and p16 deletion in 52% (54/103). Lack of p16 expression was observed in 64% (18/28) of patients. There was a good correlation between the deletion of p16 sequences and the loss of p16 expression (P = .008). Amplification of cyclin D1 had a statistically significant association with recurrence, distant metastasis, and survival at 36 months. There was a significant association between p16 deletion and the development of distant metastases. Cyclin D1 amplification and p16 deletion together correlated with recurrence, distant metastasis, and survival. We demonstrate that FISH is a simple and sensitive method for detecting cyclin D1 amplification and p16 deletion in head and neck cancer. Our results suggest that these two genetic aberrations together portend a poorer outcome than either of the abnormalities alone in head and neck cancer.

IgM myeloma: a report of four cases

IgM myeloma is a rare disease, accounting for approximately 0.5% of multiple myelomas (MM). Here we report four cases of IgM multiple myeloma. Two were diagnosed in advanced clinical stages with multiple osteolytic lesions, leading to hypercalcemia in one patient. Bone marrow morphology showed a variable degree of infiltration with mainly mature plasma cells. An immunophenotypic analysis performed in one case showed expression of CD38 and monoclonal cytoplasmatic immunoglobulin. Interphase fluorescence in situ hybridization performed in one case did not reveal any aneuploidies or deletions of the retinoblastoma, P16, or P53 tumor suppressor genes. While one patient with a smoldering IgM myeloma did not need specific therapy, the others received cytotoxic treatment based on standard chemotherapy for MM. The outcomes were one stable disease, one sustained complete remission, and one progressive disease. All four patients were alive 1 year after diagnosis. One died due to progressive disease after 31 months. We conclude that IgM myeloma shares clinical and histological features with other MM rather than with Waldenström's macroglobulinemia, which is most commonly diagnosed in cases with IgM monoclonal gammopathy. Since MM and Waldenström's macroglobulinemia differ in prognosis and treatment strategies, the two disease entities should be distinguished based on clinical criteria, bone marrow morphology, and immunophenotypic analysis.

Overexpression of pRB in human pancreatic carcinoma cells: function in chemotherapy-induced apoptosis.

Human pancreatic adenocarcinomas are highly resistant to chemotherapy. The p16 tumor-suppressor protein is inactivated in more than 90% of human pancreatic cancers. The p16 protein transcriptionally inhibits expression of retinoblastoma tumor-suppressor gene pRB. The pRB protein transcriptionally inhibits expression of the p16 gene. Because pRB normally prevents apoptosis, we investigated whether pRB is involved in resistance to chemotherapy-induced apoptosis in pancreatic cancer cells. pRB expression was examined by immunohistochemistry in 106 human pancreatic tissue specimens. The human pancreatic tumor cell line Capan-1 (pRB+/p16-) was stably transfected with p16 to functionally inactivate pRB. pRB gene expression was examined by western and northern blot analyses, and pRB function was assessed by electrophoretic mobility shift assays and promoter transactivation studies for the transcription factor E2F. Changes in cell sensitivity to chemotherapy were measured by assays for cytotoxicity and apoptosis. pRB was overexpressed in pancreatic ductal adenocarcinomas but was hardly detectable in other pancreatic malignancies, chronic pancreatitis, or nontransformed human pancreatic tissue. Expression of p16 in Capan-1 cells resulted in the loss of pRB gene and protein expression concomitant with increased activity of the transcription factor E2F, which was not detected in wild-type or control-transfected Capan-1 cells. Wild-type and control-transfected Capan-1 cells were resistant to chemotherapy-induced apoptosis, but pRB-depleted (i.e., p16-transfected) Capan-1 cells were highly sensitive. The effect was specific to pRB depletion because two other human pancreatic cancer cell lines that retained high pRB expression after p16 transfection were resistant to chemotherapy-induced apoptosis. Overexpression of pRB is associated with human pancreatic duct-cell cancer and may allow pancreatic cancer cells to evade chemotherapy-induced apoptosis.

ARF Is Not Required for Apoptosis in Rb Mutant Mouse Embryos

The retinoblastoma (RB) tumor suppressor gene occupies central roles in cell cycle control and tumor suppression [1]. Homozygous mutant (Rb−/−) embryos die at E13.5–E15.5 [2–4], exhibiting extensive apoptosis and inappropriate S phase entry in the central and peripheral nervous systems, liver, and ocular lens [2–6]. Mice simultaneously mutant for Rb and other genes can be generated to assess the requirement for these genes in cell cycle control and apoptosis. Using such analysis, E2f-1, E2f-3, p53, and Id2 have been identified as important regulators of cell cycle control and apoptosis in Rb−/− embryos [7–10]. Because unrestrained E2F activity in the absence of Rb function contributes to p53-dependent apoptosis in many systems [7, 9, 11–14], we wished to identify genes linking deregulated E2F activity to p53 activation and subsequent apoptosis. As a transcriptional target of E2F-1 [15–18], a regulator of p53 [19–21], and an important mediator of apoptosis [20–26], ARF was a strong candidate for such a role, especially since it can be upregulated in the absence of Rb[21]. From the analysis of Rb/ARF compound mutants we demonstrate that ARF is not an obligatory link between Rb inactivation and p53-dependent apoptosis.