Retinoblastoma Susceptibility Gene Rb Research Articles

Aberrant cytoplasmic accumulation of retinoblastoma protein in basal cells may lead to increased survival in malignant canine mammary tumours

The retinoblastoma susceptibility gene RB-1 is a tumour suppressor gene that encodes a protein (Rb) that regulates the transition from the G1 phase to the S phase of the cell cycle. Inactivation of the Rb gene has been shown in a variety of human tumours, including breast, ovarian, hepatic, prostatic, and endometrial carcinomas. Although Rb protein is normally expressed in the nuclei of healthy cells, during carcinogenesis there is a partial or complete loss of nuclear expression. Recently, some reports have indicated aberrant cytoplasmic expression of Rb protein. However, little is known about its cytoplasmic expression and significance as a prognostic marker in canine mammary tumours (CMT). The present study was performed on 36 malignant CMT cases in order to assess the mutational status and prognostic significance of Rb in primary malignant CMT. We report an almost complete loss of nuclear expression of Rb protein with corresponding gain of aberrant cytoplasmic expression in basal/myoepithelial cells in CMT. Strikingly, our analysis reveals a significant positive correlation between survival time and cytoplasmic expression of Rb protein in basal cells. Moreover, cytoplasmic expression of Rb protein in basal cells was also correlated with tumour grade and stage.  

The retinoblastoma protein (Rb) as an anti-apoptotic factor: expression of Rb is required for the anti-apoptotic function of BAG-1 protein in colorectal tumour cells

Although the retinoblastoma-susceptibility gene RB1 is inactivated in a wide range of human tumours, in colorectal cancer, the retinoblastoma protein (Rb) function is often preserved and the RB locus even amplified. Importantly, we have previously shown that Rb interacts with the anti-apoptotic Bcl-2 associated athanogene 1 (BAG-1) protein, which is highly expressed in colorectal carcinogenesis. Here we show for the first time that Rb expression is critical for BAG-1 anti-apoptotic activity in colorectal tumour cells. We demonstrate that Rb expression not only increases the nuclear localisation of the anti-apoptotic BAG-1 protein, but that expression of Rb is required for inhibition of apoptosis by BAG-1 both in a γ-irradiated Saos-2 osteosarcoma cell line and colorectal adenoma and carcinoma cell lines. Further, consistent with the fact that nuclear BAG-1 has previously been shown to promote cell survival through increasing nuclear factor (NF)-κB activity, we demonstrate that the ability of BAG-1 to promote NF-κB activity is significantly inhibited by repression of Rb expression. Taken together, data presented suggest a novel function for Rb, promoting cell survival through regulating the function of BAG-1. As BAG-1 is highly expressed in the majority of colorectal tumours, targeting the Rb–BAG-1 complex to promote apoptosis has exciting potential for future therapeutic development.

Transcriptional Activation by pRB and Its Coordination with SWI/SNF Recruitment

A central question in cancer biology is why most tumor susceptibility genes are linked with only limited types of cancer. Human germ-line mutation of the retinoblastoma susceptibility gene Rb1 is closely linked with just retinoblastoma and osteosarcoma, although the gene is universally expressed. Functional analysis of pRB and its close relatives, p107 and p130, has largely focused on their roles in repression of proliferation across all tissue types, but genetic evidence indicates an active requirement for pRB in osteoblast differentiation that correlates more directly with osteosarcoma susceptibility. Still, potential promoter targets of pRB and its role in normally differentiating osteoblasts remain insufficiently characterized. Here, an early marker of osteoblast differentiation, alkaline phosphatase, is identified as a direct promoter activation target of pRB. One role of pRB on this promoter is to displace the histone lysine demethylase KDM5A, thereby favoring trimethylation of H3K4, a promoter activation mark. A major new aspect of pRB-mediated transcriptional activation revealed in this promoter analysis is its role in recruitment of an activating SWI/SNF chromatin-remodeling complex. SWI/SNF is a critical coordinator of tissue-specific gene expression. In osteoblasts, SWI/SNF complexes containing the BRM ATPase repress osteoblast-specific genes to maintain the precursor state, whereas the alternative ATPase BRG1 distinguishes an activating SWI/SNF complex necessary for RNA polymerase-II recruitment. A switch from BRM to BRG1 on the alkaline phosphatase promoter marks the onset of differentiation and is accomplished in a precise two-step mechanism. Dissociation of BRM-containing SWI/SNF depends on p300, and association of BRG1-containing SWI/SNF depends on pRB.

The role of the retinoblastoma protein (Rb) in the nuclear localization of BAG-1: implications for colorectal tumour cell survival

Although the retinoblastoma susceptibility gene RB1 is inactivated in a wide variety of human cancers, the retinoblastoma protein (Rb) has been shown to be overexpressed in colon cancers, which is linked to the anti-apoptotic function of the protein. However, the mechanisms by which Rb regulates apoptosis are yet to be fully elucidated. We have established that Rb interacts with the anti-apoptotic BAG-1 (Bcl-2 associated athanogene-1) protein, and that a decrease in nuclear localization of BAG-1 is detectable when the interaction between Rb and BAG-1 is disrupted by expression of the E7 viral oncoprotein. Interestingly, although reported as deregulated in colorectal cancers, we have found that BAG-1 expression is also altered in small adenomas, where its localization was found to be predominantly nuclear. In addition, we have established that maintenance of high nuclear BAG-1 in vitro increases the resistance of adenoma-derived cells to gamma-radiation-induced apoptosis. Our work suggests a novel function for Rb, involving modulation of the subcellular localization of BAG-1. We have found predominant nuclear BAG-1 localization in small adenomas, and suggest that BAG-1 may promote colorectal tumour cell survival by making colonic epithelial cells less sensitive to DNA damage.

Loss of heterozygosity of chromosome 13 in Merkel cell carcinoma.

We have examined a series of 24 Merkel cell carcinoma (MCC) DNAs for loss of heterozygosity (LOH) at eight loci on chromosome 13. All patients were heterozygous for at least one locus. Overall, 18 of 24 (75%) patients showed LOH, among whom 10 patients demonstrated LOH at all informative loci. A single common region of loss was identified in all cases and included the marker D13S233 (13q14.3), which maps close to the retinoblastoma susceptibility gene RB1. The RB1 protein was not detected by Western blot analysis in any of nine MCC cell lines tested. These data indicate that 13q losses are the most common chromosomal losses observed to date in MCC and the likely target of these deletions is the RB1 locus.

The Retinoblastoma Susceptibility Gene RB-1 in Multiple Myeloma

Genetic mechanisms leading to the development of multiple myeloma (MM) remain poorly understood. Given the frequency of chromosome 13 deletion in MM and the localization in 13q14 of the retinoblastoma susceptibility gene RB-I, an involvement of RB-1 in MM pathogenesis has been proposed. Moreover, interleukin-6 (IL-6) has been shown to be the main growth factor for MM in vitro and in vivo. The product of the RB-I gene (pRB) can down-regdate IL-6 gene expression. Absence of pRB may then induce an autocrine IL-6 expression in myeloma cells and contribute to the autonomous growth of MM. As assessed in this review, heterozygous deletion of RB-I is very common in MM but does not alter gene transcription and protein expression. Nevertheless, homozygous deletion of RB-1 has been identified in some h4M patients with advanced disease and in the IL-6-autocrine human myeloma cell line U266. Thus, even if inactivation of RB-I appears to be only a rare and late oncogenic event in MM and is not likely to represent the main mechanism involved in IL-6 up-regulation in MM, definitive assessement of the actual role played by RB-I in MM pathogenesis still needs further investigation particularly the examination of pRB function.

Loss of heterozygosity in sporadic breast tumours at the BRCA2 locus on chromosome 13q12-q13

Loss of heterozygosity (LOH) on chromosome 13 occurs on 25-30% of breast tumours. This may reflect the inactivation of the retinoblastoma susceptibility gene RB1. However, recently another candidate tumour-suppressor gene has been identified on chromosome 13 by linkage analysis, the breast cancer susceptibility gene BRCA2. To investigate the involvement of BRCA2 in sporadic breast cancer 200 breast tumours were tested for LOH on chromosome band 13q12-q14, using 11 highly polymorphic microsatellite markers. LOH was found in 65 tumours, which all showed simultaneously loss of BRCA2 and RB1. Of 12 breast tumour cell lines tested with polymorphic microsatellite markers, seven showed a contiguous region of homozygosity on 13q12-q14, suggesting LOH in the tumour from which the cell line had been derived. One cell line showed homozygosity in the BRCA2 region and heterozygosity at RB1. This is the only indication that BRCA2 is a distinct target for LOH on chromosome 13 in addition to RB1.

Analysis of Alterations in the Retinoblastoma Gene and Tumor Grade in Bone and Soft-Tissue Sarcomas

We examined 43 sporadic bone and soft-tissue sarcomas for molecular genetic alterations affecting the retinoblastoma susceptibility gene Rb-1 (also known as RB1). The gene was altered in 6 of 14 sporadic osteosarcomas and in 5 of 29 other bone and soft-tissue sarcomas. Rb-1 messenger RNA (mRNA) transcripts were detected in normal tissues and benign lipomas, but they were absent or altered in each of the 19 sarcomas we examined. To examine the association of deletions in the Rb-1 gene with tumor grade, we correlated the DNA alterations in the Rb-1 gene with clinical data for 36 patients. The Rb-1 gene was altered in 40% of high-grade bone and soft-tissue tumors, but not in low-grade bone tumors and in only one low-grade, soft-tissue sarcoma. Overall, 10 of 25 high-grade sarcomas had detectable alterations of the Rb-1 gene compared with only 1 of 11 low-grade tumors.

Tumor Suppressor Genes: New Prospects for Cancer Research

Cancer is thought to arise from the accumulation of several genetic mutations in a single cell. Until recently, the only tumorigenic mutations that have been studied in detail are those that activate oncogenes. The discovery of tumor suppressor genes, for which inactivating mutations elicit tumorigenesis, has added a new dimension to our understanding of neoplasia. The retinoblastoma susceptibility gene RB is the prototype tumor suppressor gene and has been shown to suppress the transformed phenotype for several different cancers. Additional studies have revealed other tumor suppressor genes that may operate in a variety of tissues through a variety of mechanisms. These mechanisms may regulate the choice between cellular proliferation and differentiation and appear to involve such processes as the initiation of DNA replication, regulation of expression of certain genes, intercellular communication and adhesion, and the transduction of external signals to intracellular effectors. The elucidation of these mechanisms will enhance our understanding of both oncogenesis and the fundamental operations of the cell.

Expression and state of phosphorylation of the retinoblastoma susceptibility gene product in cycling and noncycling human hematopoietic cells.

The product of the retinoblastoma susceptibility gene RB1 (Rb) is likely to function as an inhibitor of cell growth. Previous studies have suggested that certain growth-suppressing effects of Rb are exerted in G0/G1 phase and that phosphorylation can inactivate these functions. We tested this hypothesis by examining the expression and state of phosphorylation of Rb in several lineages of primary hematopoietic cells that spontaneously arrest in G0 phase. Resting lymphocytes were found to express only unphosphorylated Rb, but phosphorylation of Rb occurred as the cells entered S phase in response to mitogens. In contrast, although monocytes and granulocytes also expressed high levels of unphosphorylated Rb, these terminally differentiated cells did not phosphorylate Rb, nor could they exit from G1 phase in response to growth factors. Thus, Rb phosphorylation appears linked to the ability of a cell to synthesize DNA. In T and B lymphocytes, Rb protein increased 8-fold after stimulation, while RB1 RNA levels increased 2- to 4-fold. Nuclear run-on assays and measurement of RB1 RNA half-life in T cells suggested that the increased RNA abundance was, at least in part, due to increased RNA stability. By contrast, Rb protein levels did not increase in either monocytes or granulocytes after stimulation, although RB1 RNA levels did increase in monocytes. Thus, there are lineage-specific differences in both the regulation of Rb phosphorylation and RB1 gene expression in lymphoid and myeloid cells.