Biomedical subjects

D Beach

Publications and source records attributed to D Beach.

At least 37 records · Page 2Linked to original sources

PubMed1997

Ran1 functions to control the Cdc10/Sct1 complex through Puc1.

We have undertaken a biochemical analysis of the regulation of the G1/S-phase transition and commitment to the cell cycle in the fission yeast Schizosaccharomyces pombe. The execution of Start requires the activity of the Cdc2 protein kinase and the Sct1/Cdc10 transcription complex. Progression through G1 also requires the Ran1 protein kinase whose inactivation leads to activation of the meiotic pathway under conditions normally inhibitory to this process. We have found that in addition to Cdc2, Sct1/Cdc10 complex formation requires Ran1. We demonstrate that the Puc1 cyclin associates with Ran1 and Cdc10 in vivo and that the Ran1 protein kinase functions to control the association between Puc1 and Cdc10. In addition, we present evidence that the phosphorylation state of Cdc10 is altered upon inactivation of Ran1. These results provide biochemical evidence that demonstrate one mechanism by which the Ran1 protein kinase serves to control cell fate through Cdc10 and Puc1.

Cell Cycle↗

PubMed1996

Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence of normal human fibroblasts.

Human diploid fibroblasts (HDFs) can be grown in culture for a finite number of population doublings before they cease proliferation and enter a growth-arrest state termed replicative senescence. The retinoblastoma gene product, Rb, expressed in these cells is hypophosphorylated. To determine a possible mechanism by which senescent human fibroblasts maintain a hypophosphorylated Rb, we examined the expression levels and interaction of the Rb kinases, CDK4 and CDK6, and the cyclin-dependent kinase inhibitors p21 and p16 in senescent HDFs. Cellular p21 protein expression increased dramatically during the final two to three passages when the majority of cells lost their growth potential and neared senescence but p21 levels declined in senescent HDFs. During this period, p16 mRNA and cellular protein levels gradually rose with the protein levels in senescent HDFs reaching nearly 40-fold higher than early passage cells. In senescent HDFs, p16 was shown to be complexed to both CDK4 and CDK6. Immunodepletion analysis of p21 and p16 from the senescent cell extracts revealed that p16 is the major CDK inhibitor for both CDK4 and CDK6 kinases. Immunoprecipitation of CDK4 and CDK6 and their associated proteins from radiolabeled extracts from senescent HDFs showed no other CDK inhibitors. Based upon these results, we propose that senescence is a multistep process requiring the expression of both p21 and p16. p16 up-regulation is a key event in the terminal stages of growth arrest in senescence, which may explain why p16 but not p21 is commonly mutated in immortal cells and human tumors.

Carrier Proteins↗

PubMed1996

A mutation in a thioredoxin reductase homolog suppresses p53-induced growth inhibition in the fission yeast Schizosaccharomyces pombe.

A strong growth inhibition is observed when the human p53 tumor suppressor gene product is expressed in the fission yeast Schizosaccharomyces pombe. This growth inhibition is specific for wild-type p53; mutant alleles of p53 derived from human tumors show a greatly decreased ability to inhibit growth. These data suggest that there may be a p53-responsive pathway in S. pombe. To identify elements in this pathway genetically, we isolated a mutant yeast strain in which the growth inhibitory activity of p53 is largely suppressed. In addition, the activity of p53 as a transcription factor is also decreased in this strain. The suppression of p53 activity is not due to a decrease in p53 expression or a failure of p53 to localize to the nucleus. This p53 suppressor mutation is in a novel S. pombe gene with homology to thioredoxin reductase genes, and has been named trr1. Strains with a mutation of, or deletion in, trr1 are sensitive to oxidizing agents, suggesting that the trr1 suppressor mutation causes partial loss of trr1 function. Since oxidizing agents are able to suppress p53 activity in vitro, this trr1 mutation may affect the activity of p53 in fission yeast by increasing the oxidation state of the tumor suppressor.

Cell Division↗

PubMed1996

Physical interaction of mammalian CDC37 with CDK4.

CDC37 was originally identified as a Start gene in budding yeast and has been shown to be required for association of CDC28 with cyclins. The exact functional mechanism by which CDC37 promotes this association, however, remains unknown. CDK4 is a cyclin D-dependent kinase that controls progression through G1 of the mammalian cell cycle. We have detected a specific association of CDK4 with the molecular chaperon HSP90 and a 44-kDa protein that we identify as mammalian CDC37. A physical interaction between CDC37 and CDK4 suggests that CDC37 may regulate the mammalian cell cycle through a direct effect on CDK4. Association of CDK4 with both CDC37 and HSP90 may also imply a mechanistic link between the functions of CDC37 and HSP90.

Amino Acid Sequence↗

PubMed1996

Cdc25 cell-cycle phosphatase as a target of c-myc.

The product of the proto-oncogene c-myc, in partnership with Max, forms a transcription factor that can promote either oncogenic transformation or apoptosis. The Myc/Max heterodimer binds to elements in the cdc25A gene and activates transcription. Like myc, cdc25A, itself a proto-oncogene, can induce apoptosis in cells depleted of growth factor, and Myc-induced apoptosis also requires cdc25A. These findings indicate that cdc25A is a physiologically relevant transcriptional target of c-myc.

3T3 Cells↗

PubMed1996

Shared role of the pRB-related p130 and p107 proteins in limb development.

The p130 protein shares extensive sequence similarity with pRB, the product of the retinoblastoma gene, and is a major E2F-associated protein in quiescent cells. To investigate its biological function, we have mutated p130 via gene targeting in the mouse. Homozygous mutation of p130 had little discernible effect on development or on the growth of mouse embryo fibroblasts in culture. Much of the E2F activity that normally associates with p130 in serum-starved mouse embryo fibroblasts associated instead with the highly related p107 protein. To determine whether p130 and p107 have overlapping biological roles, we produced mice having simultaneous inactivation of the p130 and p107 genes. Such mice exhibited deregulated chondrocyte growth, defective endochondral bone development, shortened limbs, and neonatal lethality. These findings indicate that p130 and p107 play an important role in limb development through their abilities to control chondrocyte proliferation. Thus, in certain settings p107 and p130 perform growth-regulatory functions that are not fulfilled by pRB.

Animals↗

PubMed1996

Role of the INK4a locus in tumor suppression and cell mortality.

The cell cycle inhibitor p16INK4a is inactivated in many human tumors and in families with hereditary melanoma and pancreatic cancer. Tumor-associated alterations in the INK4a locus may also affect the overlapping gene encoding p19ARF and the adjacent gene encoding p15I1NK4b, both negative regulators of cell proliferation. We report the phenotype of mice carrying a targeted deletion of the INK4a locus that eliminates both p16INK4a and p19ARF. The mice are viable but develop spontaneous tumors at an early age and are highly sensitive to carcinogenic treatments. INK4a-deficient primary fibroblasts proliferate rapidly and have a high colony-formation efficiency. In contrast with normal cells, the introduction of activated Ha-ras into INK4a-deficient fibroblasts can result in neoplastic transformation. These findings directly demonstrate that the INK4a locus functions to suppress neoplastic growth.

Alleles↗

PubMed1996

Pub1 acts as an E6-AP-like protein ubiquitiin ligase in the degradation of cdc25.

The level of the mitotic activating tyrosine phosphatase cdc25 is regulated by both transcriptional and post-transcriptional mechanisms in the fission yeast Schizosaccharomyces pombe. We have found that cdc25 is ubiquitinated and have cloned pub1, a gene which regulates this event. Pub1 contains a region highly homologous to the putative catalytic domain of the human protein ubiquitin ligase E6-AP. Disruption of pub1 elevates the level of cdc25 protein in vivo rendering cells relatively resistant to the cdc25-opposing tyrosine kinases wee1 and mik1. In addition, loss of wee1 activity in a pub1-disruption background results in a lethal premature entry into mitosis which can be rescued by loss of cdc25 function. A ubiquitin-thioester adduct of pub1 was isolated from fission yeast and disruption of pub1 dramatically reduced ubiquitination of cdc25 in vivo. These results suggest that pub1 directly ubiquitinates cdc25 in vivo.

Amino Acid Sequence↗

PubMed1996

Subcellular distribution of p21 and PCNA in normal and repair-deficient cells following DNA damage.

BACKGROUND: The p21 protein binds to both cyclin-dependent kinases (Cdks) and the proliferating cell nuclear antigen (PCNA). In mammalian cells, DNA damage results in an increase in the level of p53 protein, which stimulates expression of the gene encoding p21, which in turn leads to an inhibition of Cdk activity. Biochemical studies have shown that the direct interaction between p21 and PCNA blocks the latter's function in DNA replication but not in DNA repair. In addition to the p53-dependent damage response, the stimulation of quiescent cells with serum can also cause a p53-independent elevation in p21 gene expression. It is not clear, however, whether the induction of p21 protein under these two circumstances serves the same purpose. In this study, we have investigated the kinetics of p21 induction by DNA damage and serum stimulation and the consequent effects on cell-cycle progression. Using both normal and repair-deficient human cells, we have also analyzed the nuclear distribution of p21 in relation to that of PCNA. RESULTS: In vivo immunofluorescence staining experiments indicate that, following UV damage, DNA repair is not inhibited by the presence of a large amount of p21 protein in the nucleus; in contrast, cells undergoing DNA replication during S phase contain very low amounts of p21. The addition of serum induced a transitory elevation of p21 levels, whereas UV damage to cells resulted in a sustained, high level of p21 that was more tightly associated with the nuclear structure. Interestingly, cells deficient in global nucleotide excision-repair displayed a distinct pattern of detergent-insoluble p21 that co-localized with PCNA. CONCLUSIONS: The in vivo studies presented here, which are consistent with our previous findings in vitro, indicate that p21 has a differential effect on DNA replication and DNA repair, and that the induction of p21 by serum and DNA damage may have different consequences. Furthermore, the co-localization of p21 and PCNA in the nucleus of normal and repair-deficient human cells indicates that p21 and PCNA interact during post-damage events.

Cell Cycle↗

PubMed1996

Suppression of growth in vitro and tumorigenicity in vivo of human carcinoma cell lines by transfected p16INK4.

The function of p16INK4 as a putative tumor suppressor gene was examined by investigating its ability to inhibit the growth of cancer cell lines in vitro and tumor formation in vivo. A p16INK4 cDNA expression vector was transfected into five human cancer cell lines that varied in their p16INK4 and retinoblastoma (Rb) status. Suppression of colony-forming efficiency was seen in four cell lines. Of two cell lines wild type for p16INK4 but null for Rb protein expression, one (Hep 3B) showed inhibition of colony formation, whereas the other (Saos-2) did not. This observation may demonstrate involvement of p16INK4 independent of Rb. The transfected p16INK4 gene was frequently selected against and lost during continued growth in vitro. When compared to the colon carcinoma cell line (DLD-1),p16INK4-transfected DLD-1 clone 1 cells were less tumorigenic in athymic nude mice. Tumors arising from p16INK4-transfected DLD-1 clones, which were growth suppressed in vitro, either lost the integrated exogenous p16INK4 or expressed reduced amounts of p16INK4 protein. Therefore, p16INK4 was also selected against during tumor formation in vivo. These data are consistent with the hypothesis that p16INK4 is a tumor suppressor gene.

Animals↗

PubMed1996

Mutational analysis of the p21/WAF1/CIP1/SDI1 coding region in human tumor cell lines.

p21/WAF1/CIP1/SDI1 is an important cell-cycle mediator with tumor suppressor gene capabilities, and its inactivation could potentially lead to tumor progression. Because tumor suppressor genes are commonly inactivated by somatic and germline mutations, we analyzed a variety of human tumor cell lines for p21 mutations. We used single-strand conformational analysis and direct sequencing to identify possible mutations in the p21 coding region. Two base-alterations were observed in 41 immortalized human tumor cell lines. A previously reported polymorphism that results in a serine-to-arginine amino-acid substitution at codon 31 was found in 24% (10 of 41) of the tumor cell lines but was also found in 10% (six of 62) of normal parental DNAs tested and 7% (three of 43) of normal DNAs from patients with primary endometrial tumors. Another nucleotide substitution found at codon 80 resulted in the replacement of threonine with methionine. Codon 80 changes were found in 7% (three of 41) of the tumor cell lines (all endometrial) and in 2% (one of 62) of the normal parental DNAs. This change was not found in any of the primary endometrial tumors examined. The biological activity of these base changes was analyzed by using in vitro cyclin-dependent kinase 2-cyclin A kinase assays and calcium phosphate transfections. We observed that wild-type p21 and the p21 variants had similar growth-inhibitory abilities. Thus, our results suggest that mutation of the p21 gene is not prevalent in human tumor cell lines and is not a probable mechanism of inactivation of this gene.

Base Sequence↗

PubMed1996

A connection between pre-mRNA splicing and the cell cycle in fission yeast: cdc28+ is allelic with prp8+ and encodes an RNA-dependent ATPase/helicase.

The fission-yeast gene cdc28+ was originally identified in a screen for temperature-sensitive mutants that exhibit a cell-division cycle arrest and was found to be required for mitosis. We undertook a study of this gene to understand more fully the general requirements for entry into mitosis. Cells carrying the conditional lethal cdc28-P8 mutation divide once and arrest in G2 after being shifted to the restrictive temperature. We cloned the cdc28+ gene by complementation of the temperature-sensitive growth arrest in cdc28-P8. DNA sequence analysis indicated that cdc28+ encodes a member of the DEAH-box family of putative RNA-dependent ATPases or helicases. The Cdc28 protein is most similar to the Prp2, Prp16, and Prp22 proteins from budding yeast, which are required for the splicing of mRNA precursors. Consistent with this similarity, the cdc28-P8 mutant accumulates unspliced precursors at the restrictive temperature. Independently, we isolated a temperature-sensitive pre-mRNA splicing mutant prp8-1 that exhibits a cell-cycle phenotype identical to that of cdc28-P8. We have shown that cdc28 and prp8 are allelic. These results suggest a connection between pre-mRNA splicing and progression through the cell cycle.

Adenosine Triphosphatases↗

PubMed1996

Alteration of cell cycle kinase complexes in human papillomavirus E6- and E7-expressing fibroblasts precedes neoplastic transformation.

Expression of viral oncoproteins results in the loss of cell cycle checkpoint control and the accumulation of chromosomal abnormalities. Expression of both human papillomavirus type 16 oncoproteins, E6 and E7, in normal human fibroblasts completely dissociates p21 and proliferating cell nuclear antigen from the quarternary cyclin-cyclin-dependent kinase (CDK) complexes present in normal cells, causes disruption of the cyclin D-CDK4 complex and replacement with a CDK4-p16 complex, and leaves binary complexes of cyclin B1-CDC2 and cyclin A-CDK2 intact. These results are identical to those observed in fully transformed cells. The expression of the individual oncoproteins dramatically affects the association of proliferating cell nuclear antigen into the complexes while leaving the total cellular levels unaltered. Expression of low-risk human papillomavirus has no effect on cyclin complexes. These findings provide evidence for the gross alteration of cyclin-CDK complexes in preneoplastic cells and links this alteration to the loss of genomic stability.

Carrier Proteins↗

PubMed1996

p53-dependent association between cyclin G and the B' subunit of protein phosphatase 2A.

We and others previously showed that cyclin G is a transcriptional target of the p53 tumor suppressor protein. However, cellular proteins which might form a complex with cyclin G have not yet been identified. To gain insight into the biological role of cyclin G, we used the yeast two-hybrid screen and isolated two mouse cDNAs encoding cyclin G-interacting proteins. Interestingly, both positive cDNAs encoded B' regulatory subunits of protein phosphatase 2A (PP2A). One clone encodes B'alpha, while the other clone codes for a new member of the B' family, B'beta. B'beta is 70% identical to other members of the B' family. B'alpha associated both in vitro and in vivo with cyclin G but not with the other mammalian cyclins. Furthermore, cyclin G formed a complex with B'alpha only after induction of p53 in p53 temperature-sensitive cell lines. These results indicate that cyclin G forms a specific complex with the B' subunit of PP2A and that complex formation is regulated by p53. Potential roles for the cyclin G-B' complex in p53-mediated pathways are discussed.

Alternative Splicing↗

PubMed1995

Deletion of the p16 and p15 genes in human bladder tumors.

BACKGROUND: Two genes, p16 (also known as CDKN2, INK4A, or MTS1) and p15 (also described as INK4B or MTS2), are found in tandem at chromosome 9p21. These genes are designated as candidate tumor suppressor genes because they encode proteins that function as negative cell cycle regulators. (The encoded polypeptides inactivate specific cyclin-protein kinase complexes that are required for progression through the cell cycle.) Molecular genetic studies have revealed that deletion of the p16 and p15 genes occurs frequently in cancer cell lines and in certain malignant neoplasms. PURPOSE: We evaluated the frequency of p16 and p15 gene alterations in a well-characterized cohort of human transitional cell bladder cancers, and we explored potential associations between alterations in these genes and tumor stage and/or grade. METHODS: Tumor tissue and normal tissue from 110 patients with transitional cell carcinoma of the urinary bladder were examined. The status of the p16 and p15 genes in these tissues was determined by Southern blotting and hybridization with gene-specific probes, by coupled polymerase chain reaction and single-strand conformation polymorphism analysis (PCR-SSCP), and by sequencing DNA fragments produced during PCR. Associations between alterations in the genes and tumor stage and/or grade were evaluated using the two-tailed Fisher's exact test. RESULTS: Homozygous deletion (both alleles lost) of the p16 and the p15 genes was observed in 11 and nine bladder tumors, respectively. Eight of the 11 tumors exhibiting complete loss of the p16 gene also displayed homozygous deletion of the p15 gene. Exclusive loss of either gene was detected in only three tumors. Hemizygous deletion (one allele lost, also referred to as loss of heterozygosity [LOH] of the p16 and/or p15 genes was observed in eight tumors. Rearrangement of the two genes was indicated in three additional tumors. No point mutations were identified in either gene. The overall frequency of alteration in this cohort of bladder tumors was approximately 18% for each gene (in 20 [18.3%, 95% confidence interval (CI) = 11.1%-25.6%] of 109 informative tumors for the p16 gene and in 18 [18%, 95% CI = 10.5%-25.5%] of 100 informative tumors for the p15 gene). A statistically significant association between p16 gene alteration and bladder tumors of low stage (P < .01) and grade (P < .01) was observed; a significant association between p15 gene alteration and tumors of low stage (P < .01) was also detected. CONCLUSIONS: Alteration of the p16 and p15 genes, especially coincident homozygous deletion, appears to be a common event in bladder cancer.

Aged↗

PubMed1995

Radiation-induced cell cycle arrest compromised by p21 deficiency.

The protein p21 is a dual inhibitor of cyclin-dependent kinases and proliferating-cell nuclear antigen (PCNA), both of which are required for passage through the cell cycle. The p21 gene is under the transcriptional control of p53 (ref. 5), suggesting that p21 might promote p53-dependent cell cycle arrest or apoptosis. p21 has also been implicated in cell senescence and in cell-cycle withdrawal upon terminal differentiation. Here we investigate the role of p21 in these processes using chimaeric mice composed partly of p21-/- and partly of p21+/+ cells. Immunohistochemical studies of the p21+/+ and p21-/- components of adult small intestine indicated that deletion of p21 has no detectable effect on the migration-associated differentiation of the four principal intestinal epithelial cell lineages or on p53-dependent apoptosis following irradiation. However, p21-/- mouse embryo fibroblasts are impaired in their ability to undergo G1 arrest following DNA damage.

Animals↗

PubMed1995

p19Skp1 and p45Skp2 are essential elements of the cyclin A-CDK2 S phase kinase.

In normal human fibroblasts, cyclin A-CDK2 exists in a quaternary complex that contains p21 and PCNA. In many transformed cells, p21 disappears, and a substantial fraction of cyclin A-CDK2 complexes with p9CKS1/CKS2, p19, and p45. To investigate the significance of these rearrangements, we have isolated cDNAs encoding p19 and p45. In vitro reconstitution demonstrated that binding of p19 to cyclin A-CDK2 requires p45. Addition of these proteins to the kinase had no substantial effect on the kinase activity in vitro. Interference with p45 function in vivo by microinjection of antibodies or antisense oligonucleotides prevented entry into S phase in both normal and transformed cells. Cyclin A-CDK2 has previously been identified as a kinase whose activity is essential for S phase. Our results identify p45 as an essential element of this activity. The abundance of p45 is greatly increased in many transformed cells. This could result in changes in cell cycle control that contribute to the process of cellular transformation.

Amino Acid Sequence↗

PubMed1995

CDC25 phosphatases as potential human oncogenes.

Cyclin-dependent kinases (CDKs) are activated by CDC25 phosphatases, which remove inhibitory phosphate from tyrosine and threonine residues. In human cells, CDC25 proteins are encoded by a multigene family, consisting of CDC25A, CDC25B, and CDC25C. In rodent cells, human CDC25A or CDC25B but not CDC25C phosphatases cooperate with either Ha-RASG12V or loss of RB1 in oncogenic focus formation. Such transformants were highly aneuploid, grew in soft agar, and formed high-grade tumors in nude mice. Overexpression of CDC25B was detected in 32 percent of human primary breast cancers tested. The CDC25 phosphatases may contribute to the development of human cancer.

Animals↗