Biomedical subjects

J DeGregori

Publications and source records attributed to J DeGregori.

27 records · Page 2Linked to original sources

PubMed1996

Inhibition of cyclin D-CDK4/CDK6 activity is associated with an E2F-mediated induction of cyclin kinase inhibitor activity.

Alterations of various components of the cell cycle regulatory machinery that controls the progression of cells from a quiescent to a growing state contribute to the development of many human cancers. Such alterations include the deregulated expression of G1 cyclins, the loss of function of activities such as those of protein p16INK4a that control G1 cyclin-dependent kinase activity, and the loss of function of the retinoblastoma protein (RB), which is normally regulated by the G1 cyclin-dependent kinases. Various studies have revealed an inverse relationship in the expression of p16INK4a protein and the presence of functional RB in many cell lines. In this study we show that p16INK4a is expressed in cervical cancer cell lines in which the RB gene, Rb, is not functional, either as a consequence of Rb mutation or expression of the human papillomavirus E7 protein. We also demonstrate that p16INK4a levels are increased in primary cells in which RB has been inactivated by DNA tumor virus proteins. Given the role of RB in controlling E2F transcription factor activity, we investigated the role of E2F in controlling p16INK4a expression. We found that E2F1 overexpression leads to an inhibition of cyclin D1-dependent kinase activity and induces the expression of a p16-related transcript. We conclude that the accumulation of G1 cyclin-dependent kinase activity during normal G1 progression leads to E2F accumulation through the inactivation of RB, and that this then leads to the induction of cyclin kinase inhibitor activity and a shutdown of G1 kinase activity.

Animals↗

PubMed1996

The accumulation of an E2F-p130 transcriptional repressor distinguishes a G0 cell state from a G1 cell state.

Previous studies have demonstrated cell cycle-dependent specificities in the interactions of E2F proteins with Rb family members. We now show that the formation of an E2F-p130 complex is unique to cells in a quiescent, G0 state. The E2F-p130 complex does not reform when cells reenter a proliferative state and cycle through G1. The presence of an E2F-p130 complex in quiescent cells coincides with the E2F-mediated repression of transcription of the E2F1 gene, and we show that the E2F sites in the E2F1 promoter are important as cells enter quiescence but play no apparent role in cycling cells. In addition, the decay of the E2F-p130 complex as cells reenter the cell cycle requires the action of G1 cyclin-dependent kinase activity. We conclude that the accumulation of the E2F-p130 complex in quiescent cells provides a negative control of certain key target genes and defines a functional distinction between these G0 cells and cells that exist transiently in G1.

3T3 Cells↗

PubMed1996

Expression of the HsOrc1 gene, a human ORC1 homolog, is regulated by cell proliferation via the E2F transcription factor.

The initiation of DNA replication in Saccharomyces cerevisiae requires the action of a multisubunit complex of six proteins known as the origin recognition complex (ORC). The identification of higher eukaryotic homologs of several ORC components suggests a universal role for this complex in DNA replication. We now demonstrate that the expression of one of these homologs is regulated by cell proliferation. Expression of the human Orc1 gene (HsOrc1) is low in quiescent cells, and it is then dramatically induced upon stimulation of cell growth. In contrast, expression of the HsOrc2 gene does not appear to be similarly regulated. We have isolated the promoter that regulates HsOrc1 transcription, and we show that the promoter confers cell growth-dependent expression. We also demonstrate that the cell growth control is largely the consequence of E2F-dependent negative transcription control in quiescent cells. Activation of HsOrc1 transcription following growth stimulation requires G1 cyclin-dependent kinase activity, and forced E2F1 expression can bypass this requirement. These results thus provide a direct link between the initiation of DNA replication and the cell growth regulatory pathway involving G1 cyclin-dependent kinases, the Rb tumor suppressor, and E2F.

Base Sequence↗

PubMed1995

Regulation of the cyclin E gene by transcription factor E2F1.

A variety of results point to the transcription factor E2F as a critical determinant of the G1/S-phase transition during the cell cycle in mammalian cells, serving to activate the transcription of a group of genes that encode proteins necessary for DNA replication. In addition, E2F activity appears to be directly regulated by the action of retinoblastoma protein (RB) and RB-related proteins and indirectly regulated through the action of G1 cyclins and associated kinases. We now show that the accumulation of G1 cyclins is regulated by E2F1. E2F binding sites are found in both the cyclin E and cyclin D1 promoters, both promoters are activated by E2F gene products, and at least for cyclin E, the E2F sites contribute to cell cycle-dependent control. Most important, the endogenous cyclin E gene is activated following expression of the E2F1 product encoded by a recombinant adenovirus vector. These results suggest the involvement of E2F1 and cyclin E in an autoregulatory loop that governs the accumulation of critical activities affecting the progression of cells through G1.

Animals↗

PubMed1995

E2F-1 accumulation bypasses a G1 arrest resulting from the inhibition of G1 cyclin-dependent kinase activity.

Numerous experiments have defined a critical role for the G1 cyclins and associated kinases in allowing a normal progression of cells from a quiescent state, through G1, and into S phase. We now demonstrate that G1 cyclin-dependent kinase activity is critical for the accumulation of E2F activity late in G1. Moreover, E2F-1 overexpression can overcome a G1 arrest caused by the inhibition of G1 cyclin-dependent kinase activity, consistent with E2F activation being an important consequence of the action of G1 cyclins. E2F-1 also overcomes a G1 block caused by gamma irradiation and leads to an apparent complete replication of the cellular genome and entry into mitosis. This E2F-1-mediated induction of S phase and mitosis is not accompanied by the rise in either cyclin D-associated kinase activity or cdk2 activity that is normally observed during the G1 phase of the cell cycle. We conclude that one key function for G1 cyclin-dependent kinase activity is the activation of E2F-1, that the accumulation of E2F activity may be sufficient to allow initiation and completion of S phase, but that additional events, including G1 cyclin kinase activity, are likely necessary for a normal proliferative event.

Blotting, Western↗

PubMed1995

E2F1 overexpression in quiescent fibroblasts leads to induction of cellular DNA synthesis and apoptosis.

Various experiments have demonstrated a role for the E2F transcription factor in the regulation of cell growth during the G0/G1/S phase transition. Indeed, overexpression of the E2F1 product, a component of the cellular E2F activity, induces DNA synthesis in quiescent fibroblasts. To provide an approach to a more detailed biochemical analysis of these events, we have made use of a recombinant adenovirus containing the E2F1 cDNA in order to efficiently express the E2F1 product in an entire population of cells. We demonstrate an induction of DNA synthesis when quiescent cells are infected with the E2F1 recombinant virus. However, we also find that the induction does not lead to a complete replication of the cellular genome, as revealed by flow cytometry. The incomplete nature of cellular DNA replication is due, at least in part, to the fact that E2F1 overexpression leads to massive cell death that is characteristic of apoptosis. This E2F1-mediated induction of apoptosis is largely dependent on endogenous wild-type p53 activity and can be subverted by introducing mutant forms of p53 into these cells or by overexpressing E2F1 in fibroblasts derived from p53-null mouse embryos. We conclude that E2F1 can induce events leading to S phase but that the process is not normal and appears to result from the activation of a cell death pathway.

Adenoviridae↗

PubMed1995

Cellular targets for activation by the E2F1 transcription factor include DNA synthesis- and G1/S-regulatory genes.

Although a number of transfection experiments have suggested potential targets for the action of the E2F1 transcription factor, as is the case for many transcriptional regulatory proteins, the actual targets in their normal chromosomal environment have not been demonstrated. We have made use of a recombinant adenovirus containing the E2F1 cDNA to infect quiescent cells and then measure the activation of endogenous cellular genes as a consequence of E2F1 production. We find that many of the genes encoding S-phase-acting proteins previously suspected to be E2F targets, including DNA polymerase alpha, thymidylate synthase, proliferating cell nuclear antigen, and ribonucleotide reductase, are indeed induced by E2F1. Several other candidates, including the dihydrofolate reductase and thymidine kinase genes, were only minimally induced by E2F1. In addition to the S-phase genes, we also find that several genes believed to play regulatory roles in cell cycle progression, such as the cdc2, cyclin A, and B-myb genes, are also induced by E2F1. Moreover, the cyclin E gene is strongly induced by E2F1, thus defining an autoregulatory circuit since cyclin E-dependent kinase activity can stimulate E2F1 transcription, likely through the phosphorylation and inactivation of Rb and Rb family members. Finally, we also demonstrate that a G1 arrest brought about by gamma irradiation is overcome by the overexpression of E2F1 and that this coincides with the enhanced activation of key target genes, including the cyclin A and cyclin E genes.

Adenoviridae↗

PubMed1994

A murine homolog of the yeast RNA1 gene is required for postimplantation development.

A gene has been characterized that is required for postimplantation mouse development. The gene, designated fug1, was disrupted in embryonic stem cells by the U3Neo gene trap retrovirus, and the disrupted allele was introduced into the germ line. Homozygous mutant embryos arrest at the egg cylinder stage at about embryonic day 6 and are mostly resorbed by day 8.5. The appearance of the proamniotic cavity is delayed, and epiblast cells that surround the cavity are disorganized. fug1 transcripts are undetectable at E6 but are induced throughout the embryo after E6.5. The gene is expressed at low levels in all adult tissues examined, maps to chromosome 15, and is conserved among mammals. The cDNA sequence encodes a protein of 589 amino acids, the first 400 of which are 38% identical to the Saccaromyces cerevisiae RNA1 gene. Regions of greatest similarity include a long acidic domain and 11 leucine-rich motifs, thought to mediate high affinity protein-protein interactions. These similarities suggest that Fug1 may be required for developmental changes in RNA processing or chromatin structure prior to gastrulation.

Amino Acid Sequence↗

PubMed1991

Mechanisms of oncogene cooperation: activation and inactivation of a growth antagonist.

Gene transfer experiments have defined limitations with regard to the ability of individual oncogenes to transform cultured cells to a tumorigenic state. The stable transformation of REF52 cells by either the ras or sis oncogenes requires the continuous expression of a second collaborating oncogene, such as adenovirus-5 E1A or SV40 large T-antigen. Our studies suggest that the function of the nuclear collaborators is to antagonize dominant growth controls which limit the ability of REF52 cells to proliferate in response to mitogenic stimuli.

Adenovirus Early Proteins↗