Whole chromosome instability resulting from the synergistic effects of pRB and p53 inactivation.

Whole chromosome instability (CIN) is a common feature of cancer cells and has been linked to increased tumor evolution and metastasis. Several studies have shown that the loss of the pRB tumor suppressor causes mitotic defects and chromosome mis-segregation. pRB is inactivated in many types of cancer and this raises the possibility that the loss of pRB may be a general cause of CIN in tumors. Paradoxically, retinoblastoma tumor cells have a relatively stable karyotype and currently the circumstances in which pRB inactivation causes CIN in human cancers are unclear. Here we utilize a fluorescence in situ hybridization-based approach to score numerical heterogeneity in chromosome copy number as a readout of CIN. Using this technique, we show that high levels of CIN correlate with the combined inactivation of pRB and p53 and that this association is evident in two independent panels of cancer cell lines. Retinoblastoma cell lines characteristically retain a wild-type TP53 gene, providing an opportunity to test the relevance of this functional relationship. We show that retinoblastoma cell lines display mitotic defects similar to those seen when pRB is depleted from non-transformed cells, but that the presence of wild-type p53 suppresses the accumulation of aneuploid cells. A similar synergy between pRB and p53 inactivation was observed in HCT116 cells. These results suggest that the loss of pRB promotes segregation errors, whereas loss of p53 allows tolerance and continued proliferation of the resulting, genomically unstable cancer cells. Hence, it is the cooperative effect of inactivation of both pRB and p53 tumor suppressor pathways that promotes CIN.

Functional antagonism between E2F family members.

E2F is a heterogenous transcription factor and its role in cell cycle control results from the integrated activities of many different E2F family members. Unlike mammalian cells, that have a large number of E2F-related genes, the Drosophila genome encodes just two E2F genes, de2f1 and de2f2. Here we show that de2f1 and de2f2 provide different elements of E2F regulation and that they have opposing functions during Drosophila development. dE2F1 and dE2F2 both heterodimerize with dDP and bind to the promoters of E2F-regulated genes in vivo. dE2F1 is a potent activator of transcription, and the loss of de2f1 results in the reduced expression of E2F-regulated genes. In contrast, dE2F2 represses the transcription of E2F reporters and the loss of de2f2 function results in increased and expanded patterns of gene expression. The loss of de2f1 function has previously been reported to compromise cell proliferation. de2f1 mutant embryos have reduced expression of E2F-regulated genes, low levels of DNA synthesis, and hatch to give slow-growing larvae. We find that these defects are due in large part to the unchecked activity of dE2F2, since they can be suppressed by mutation of de2f2. Examination of eye discs from de2f1; de2f2 double-mutant animals reveals that relatively normal patterns of DNA synthesis can occur in the absence of both E2F proteins. This study shows how repressor and activator E2Fs are used to pattern transcription and how the net effect of E2F on cell proliferation results from the interplay between two types of E2F complexes that have antagonistic functions.

Prognostic and pathologic significance of quantitative protein expression profiling in human gliomas.

PURPOSE: Analysis of tumor-derived genetic lesions has provided insights into molecular pathogenesis of human gliomas. Because these changes represent only one of several mechanisms that alter gene expression during tumorigenesis, it is likely that further information will be obtained from a careful analysis of important regulatory proteins present in these tumors. EXPERIMENTAL DESIGN: We have quantified the levels of key cell cycle/signaling proteins in 94 prospectively collected, meticulously preserved, "snap frozen" glioma specimens and have compared these levels with histopathological data and patient outcome. RESULTS: The results of these experiments confirm that the levels of wild-type tumor suppressor proteins, such as p53, pRB, PTEN, p14(ARF), and p16(INK4), are lost or severely reduced in most gliomas, and that epidermal growth factor receptor, 2human telomerase reverse transcriptase, and cyclin-dependent kinase 4 are overexpressed frequently and with a few exceptions, almost exclusively, in glioblastomas. In addition, we report frequent underexpression of E2F-1 (in 55% of gliomas) and cyclin E overexpression (in 26% of gliomas), which have not yet been reported on the genomic level. Several of these markers significantly correlated with histopathological grade, and the levels of five proteins showed significant association with patient outcome. In particular, overexpression of epidermal growth factor receptor, human telomerase reverse transcriptase, cyclin-dependent kinase 4, and cyclin E was largely restricted to glioblastomas and was significantly associated with reduced patient survivals. CONCLUSIONS: We conclude that the quantitation of cell cycle/signaling proteins from meticulously preserved glioma specimens provides further insights into the molecular pathogenesis of human gliomas and yields valuable prognostic information.

Cyclin-dependent kinases and P53 pathways are activated independently and mediate Bax activation in neurons after DNA damage.

DNA damage has been implicated as one important initiator of cell death in neuropathological conditions such as stroke. Accordingly, it is important to understand the signaling processes that control neuronal death induced by this stimulus. Previous evidence has shown that the death of embryonic cortical neurons treated with the DNA-damaging agent camptothecin is dependent on the tumor suppressor p53 and cyclin-dependent kinase (CDK) activity and that the inhibition of either pathway alone leads to enhanced and prolonged survival. We presently show that p53 and CDKs are activated independently on parallel pathways. An increase in p53 protein levels, nuclear localization, and DNA binding that result from DNA damage are not affected by the inhibition of CDK activity. Conversely, no decrease in retinoblastoma protein (pRb) phosphorylation was observed in p53-deficient neurons that were treated with camptothecin. However, either p53 deficiency or the inhibition of CDK activity alone inhibited Bax translocation, cytochrome c release, and caspase-3-like activation. Taken together, our results indicate that p53 and CDK are activated independently and then act in concert to control Bax-mediated apoptosis.

Retinoblastoma protein partners.

Studies of the retinoblastoma gene (Rb) have shown that its protein product (pRb) acts to restrict cell proliferation, inhibit apoptosis, and promote cell differentiation. The frequent mutation of the Rb gene, and the functional inactivation of pRb in tumor cells, have spurred interest in the mechanism of pRb action. Recently, much attention has focused on pRb's role in the regulation of the E2F transcription factor. However, biochemical studies have suggested that E2F is only one of many pRb-targets and, to date, at least 110 cellular proteins have been reported to associate with pRb. The plethora of pRb-binding proteins raises several important questions. How many functions does pRb possess, which of these functions are important for development, and which contribute to tumor suppression? The goal of this review is to summarize the current literature of pRb-associated proteins.

Mutagenesis of the pRB pocket reveals that cell cycle arrest functions are separable from binding to viral oncoproteins.

The pocket domain of pRB is required for pRB to arrest the cell cycle. This domain was originally defined as the region of the protein that is necessary and sufficient for pRB's interaction with adenovirus E1A and simian virus s40 large T antigen. These oncoproteins, and other pRB-binding proteins that are encoded by a variety of plant and animal viruses, use a conserved LXCXE motif to interact with pRB. Similar sequences have been identified in multiple cellular pRB-binding proteins, suggesting that the viruses have evolved to target a highly conserved binding site of pRB that is critical for its function. Here we have constructed a panel of pRB mutants in which conserved amino acids that are predicted to make close contacts with an LXCXE peptide were altered. Despite the conservation of the LXCXE binding site throughout evolution, pRB mutants that lack this site are able to induce a cell cycle arrest in a pRB-deficient tumor cell line. This G(1) arrest is overcome by cyclin D-cdk4 complexes but is resistant to inactivation by E7. Consequently, mutants lacking the LXCXE binding site were able to induce a G(1) arrest in HeLa cells despite the expression of HPV-18 E7. pRB mutants lacking the LXCXE binding site are defective in binding to adenovirus E1A and human papillomavirus type 16 E7 protein but exhibit wild-type binding to E2F or DP, and they retain the ability to interact with CtIP and HDAC1, two transcriptional corepressors that contain LXCXE-like sequences. Consistent with these observations, the pRB mutants are able to actively repress transcription. These observations suggest that viral oncoproteins depend on the LXCXE-binding site of pRB for interaction to a far greater extent than cellular proteins that are critical for cell cycle arrest or transcriptional repression. Mutation of this binding site allows pRB to function as a cell cycle regulator while being resistant to inactivation by viral oncoproteins.

Disruption of retinoblastoma protein family function by human papillomavirus type 16 E7 oncoprotein inhibits lens development in part through E2F-1.

Complexes between the retinoblastoma protein (pRb) and the transcription factor E2F-1 are thought to be important for regulating cell proliferation. We have shown previously that the E7 oncoprotein from human papillomavirus type 16, dependent upon its binding to pRb proteins, induces proliferation, disrupts differentiation, and induces apoptosis when expressed in the differentiating, or fiber, cells of the ocular lenses in transgenic mice. Mice that carry a null mutation in E2F-1 do not exhibit any defects in proliferation and differentiation in the lens. By examining the lens phenotype in mice that express E7 on an E2F-1 null background, we now show genetic evidence that E7's ability to alter the fate of fiber cells is partially dependent on E2F-1. On the other hand, E2F-1 status does not affect E7-induced proliferation in the undifferentiated lens epithelium. These data provide genetic evidence that E2F-1, while dispensible for normal fiber cell differentiation, is one mediator of E7's activity in vivo and that the requirement for E2F-1 is context dependent. These data suggest that an important role for pRb-E2F-1 complex during fiber cell differentiation is to negatively regulate cell cycle progression, thereby allowing completion of the differentiation program to occur.

Key roles for E2F1 in signaling p53-dependent apoptosis and in cell division within developing tumors.

Apoptosis induced by the p53 tumor suppressor can attenuate cancer growth in preclinical animal models. Inactivation of the pRb proteins in mouse brain epithelium by the T121 oncogene induces aberrant proliferation and p53-dependent apoptosis. p53 inactivation causes aggressive tumor growth due to an 85% reduction in apoptosis. Here, we show that E2F1 signals p53-dependent apoptosis since E2F1 deficiency causes an 80% apoptosis reduction. E2F1 acts upstream of p53 since transcriptional activation of p53 target genes is also impaired. Yet, E2F1 deficiency does not accelerate tumor growth. Unlike normal cells, tumor cell proliferation is impaired without E2F1, counterbalancing the effect of apoptosis reduction. These studies may explain the apparent paradox that E2F1 can act as both an oncogene and a tumor suppressor in experimental systems.

Loss of E2F-1 reduces tumorigenesis and extends the lifespan of Rb1(+/-)mice.

Mutation of the retinoblastoma tumour-suppressor gene (RB) leads to the deregulation of many proteins and transcription factors that interact with the retinoblastoma gene product (pRB), including members of the E2F transcription factor family. As pRB is known to repress E2F transcriptional activity and overexpression of E2F is sufficient for cell cycle progression, it is thought that pRB suppresses growth in part by repressing E2F-mediated transcription. Previously, we reported that loss of E2f1 in mice results in tissue-specific tumour induction and tissue atrophy, demonstrating that E2F-1 normally controls growth both positively and negatively in a tissue-specific fashion. To determine whether E2F-1 deregulation--as a result of loss of pRB--promotes proliferation in vivo, we have tested whether loss of E2f1 interferes with the pituitary and thyroid tumorigenesis that occurs in Rb1(+/-) mice. We have found that loss of E2f1 reduces the frequency of pituitary and thyroid tumours, and greatly lengthens the lifespan of Rb1(+/-); E2f1(-/-) animals, demonstrating that E2F-1 is an important downstream target of pRB during tumorigenesis. Furthermore, loss of E2f1 reduces a previously reported strain-dependent difference in Rb1(+/-) lifespan, suggesting that E2f1 or an E2F-1-regulated gene acts as a genetic modifier between the 129/Sv and C57BL/6 strains.

Tumor induction and tissue atrophy in mice lacking E2F-1.

The retinoblastoma tumor suppressor protein (pRB) is a transcriptional repressor that regulates gene expression by physically associating with transcription factors such as E2F family members. Although pRB and its upstream regulators are commonly mutated in human cancer, the physiological role of the pRB-E2F pathway is unknown. To address the function of E2F-1 and pRB/E2F-1 complexes in vivo, we have produced mice homozygous for a nonfunctional E2F-1 allele. Mice lacking E2F-1 are viable and fertile, yet experience testicular atrophy and exocrine gland dysplasia. Surprisingly, mice lacking E2F-1 develop a broad and unusual spectrum of tumors. Although overexpression of E2F-1 in tissue culture cells can stimulate cell proliferation and be oncogenic, loss of E2F-1 in mice results in tumorigenesis, demonstrating that E2F-1 also functions as a tumor suppressor.

Processing of mitochondrial RNA in Aspergillus nidulans.

Genes for cytochrome oxidase subunit I (oxiA), ATPase subunit 9, NADH dehydrogenase subunit 3 (ndhC) and cytochrome oxidase subunit II (oxiB) are located within a 7.2 kb (1 kb = 10(3) bases or base-pairs) segment of the Aspergillus nidulans mitochondrial genome. Northern hybridization shows that abundant RNA molecules of 4.0, 2.5 and 1.5 kb, each containing copies of two or more genes, are transcribed from this region. The 4.0 kb molecule, which contains copies of each of the four genes but lacks the three oxiA introns, is cleaved at a point just upstream from ndhC to give rise to the 2.5 kb RNA, which contains copies of oxiA and the ATPase subunit 9 gene, and the 1.5 kb RNA, which carries ndhC and oxiB. The ATPase subunit 9 gene, which has no identified function, is therefore transcribed into an abundant RNA. S1 nuclease analysis indicates that there are no additional introns in the amino-terminal region of oxiA and that the 4.0 and 2.5 kb transcripts of this gene have staggered 5' termini, the most upstream of which is adjacent to the 3' end of the histidinyl-tRNA gene. The results suggest that transcription of this genome proceeds via a very limited number of primary transcripts with mature RNAs produced by extensive processing events including tRNA excision. RNA synthesis and processing in A. nidulans mitochondria therefore resembles the events occurring in metazoa rather than yeast.

The mitochondrial ribosomal RNA molecules of Aspergillus nidulans.

The 16S and 23S mitochondrial rRNAs of Aspergillus nidulans have been identified by Northern hybridisation and the ends of the molecules mapped onto the mitochondrial genome by S1 nuclease analysis. The results show that both the rRNA molecules are longer than originally reported, forcing a reassessment of the potential secondary structures that can form in the terminal regions. In particular, structures resembling the 5.8S- and 4.5S-like domains of the bacterial large rRNA can now be recognised within the A. nidulans 23S molecule. The new 5' termini of the 16S and 23S genes lie within conserved 18-bp sequences that may be promoters but are more likely to be processing signals that cleave the mature rRNAs from larger precursor molecules. The new end of the 23S gene abuts the 5' end of the threonine-tRNA gene.