Try a new search

Format these results:

Searched for:

in-biosketch:yes

person:paganm02

Total Results:

260


Ubiqfitin-dependent degradation of cyclin b is accelerated during an endomitotic cell cycle

Zhanj, V; Wang, Z; Pagano, M; Rawid, K
Endomitosis mWohcs DNA replicalion in the absence ol propei mitosis and cytokinesis. We found that during the endomilolic cell cWclc ol tWo Jilicrcnl mcgakaiyocylic cell lines, the lcW'cls of CWclm B l protein and the actiWitW ol Cdc2, the CWclm Bl dcpcndcnl kinasc. were reduced as compared to mcgakaryocytcs undergoing a mitolic cell cycle. In contrast, the levels ol CWclm A. another Cdc2-associatcd cyclin, Wcrc compaiahle dunng both cell cycles. The expression of cyclin Bl nrRNA was c-qnnalent m piohlcralmg and pohploidi/mg cells, bul the rate ol Cyclin Bl protein degradation ""'as enhanced in polWploidiJ.mg mcgakaryocWtes. These Undings lead us lo lurthcr mWcstigatc whether the ubiquilinprotcasomc palhuay icsponsiblc lor CWchn B degradation is accelerated in polyploid mcgakaiyocylcs Our data indicated that polWploidi/mg megakaiyoc-ytic cell lines and pnmary bone manoW mcgakaiWocWlcs display an increased activity o! the ubiquitin-prolcasomc pathvWaW Which dcgiadcs CWclm BI, as compared It) proh I crating mcgakaiyocWlic cell lines or to bone manou cells, icspcclncly. This dcgiadation had all the hallmarks ol a ubiquitm palhwaj, including the dependent) on ATI', inhibition by apWrasc. and the appealance of high molecular ucighl conjupalcd lorms ol Cyclin Bl. The increased potential ol polyploid nrcgakaty ocW tes to degrade CAchn Bl maW be part ol cellular progiamming u Inch leads li > aborted mitosis
SCOPUS:33750157713
ISSN: 0892-6638
CID: 589812

Cell cycle control and cancer [Review]

Draetta, G; Pagano, M
ISI:A1996BG58V00025
ISSN: 0065-7743
CID: 98376

Cell cycle and cancer: critical events at the G1 restriction point

DelSal, G; Loda, M; Pagano, M
In eukaryotic cells, each phase of the cell division cycle is controlled by the sequential activation of various cyclin-dependent kinases (Cdks). These kinases are known to phosphorylate various substrates whose activity is critical for cell cycle progression. As key regulators of the cell cycle, Cdks must be strictly controlled by both extracellular and intracellular signals for adequate responses to occur. There are several distinct molecular mechanisms for controlling the activity of the different Cdks: regulated synthesis and destruction of the activating subunit (cyclin), regulated synthesis and destruction of the inhibitory subunit (Cki), and posttranslational modification of the kinase subunit by highly specific kinases and phosphatases. During the G1 phase of the cell cycle, cells sense, integrate positive and negative signals, and transmit them to the cell cycle machinery. Because of this pivotal role, a vast majority of oncogenic events selectively target elements controlling the G1. In this review we discuss the elements controlling the G1 phase in relationship to the genesis of cancer.
PMID: 9109501
ISSN: 0893-9675
CID: 539962

Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27

Pagano M; Tam SW; Theodoras AM; Beer-Romero P; Del Sal G; Chau V; Yew PR; Draetta GF; Rolfe M
The p27 mammalian cell cycle protein is an inhibitor of cyclin-dependent kinases. Both in vivo and in vitro, p27 was found to be degraded by the ubiquitin-proteasome pathway. The human ubiquitin-conjugating enzymes Ubc2 and Ubc3 were specifically involved in the ubiquitination of p27. Compared with proliferating cells, quiescent cells exhibited a smaller amount of p27 ubiquitinating activity, which accounted for the marked increase of p27 half-life measured in these cells. Thus, the abundance of p27 in cells is regulated by degradation. The specific proteolysis of p27 may represent a mechanism for regulating the activity of cyclin-dependent kinases
PMID: 7624798
ISSN: 0036-8075
CID: 21099

Human cyclin E, a nuclear protein essential for the G1-to-S phase transition

Ohtsubo M; Theodoras AM; Schumacher J; Roberts JM; Pagano M
Cyclin E was first identified by screening human cDNA libraries for genes that would complement G1 cyclin mutations in Saccharomyces cerevisiae and has subsequently been found to have specific biochemical and physiological properties that are consistent with it performing a G1 function in mammalian cells. Most significantly, the cyclin E-Cdk2 complex is maximally active at the G1/S transition, and overexpression of cyclin E decreases the time it takes the cell to complete G1 and enter S phase. We have now found that mammalian cells express two forms of cyclin E protein which differ from each other by the presence or absence of a 15-amino-acid amino-terminal domain. These proteins are encoded by alternatively spliced mRNAs and are localized to the nucleus during late G1 and early S phase. Fibroblasts engineered to constitutively overexpress either form of cyclin E showed elevated cyclin E-dependent kinase activity and a shortened G1 phase of the cell cycle. The overexpressed cyclin E protein was detected in the nucleus during all cell cycle phases, including G0. Although the cyclin E protein could be overexpressed in quiescent cells, the cyclin E-Cdk2 complex was inactive. It was not activated until 6 to 8 h after readdition of serum, 4 h earlier than the endogenous cyclin E-Cdk2. This premature activation of cyclin E-Cdk2 was consistent with the extent of G1 shortening caused by cyclin E overexpression. Microinjection of affinity-purified anti-cyclin E antibodies during G1 inhibited entry into S phase, whereas microinjection performed near the G1/S transition was ineffective. These results demonstrate that cyclin E is necessary for entry into S phase. Moreover, we found that cyclin E, in contrast to cyclin D1, was required for the G1/S transition even in cells lacking retinoblastoma protein function. Therefore, cyclins E and D1 control two different transitions within the human cell cycle
PMCID:230491
PMID: 7739542
ISSN: 0270-7306
CID: 21100

Reconstitution of p53-ubiquitinylation reactions from purified components: the role of human ubiquitin-conjugating enzyme UBC4 and E6-associated protein (E6AP)

Rolfe, M; Beer-Romero, P; Glass, S; Eckstein, J; Berdo, I; Theodoras, A; Pagano, M; Draetta, G
The E6 protein of the high-risk human papillomaviruses inactivates the tumor suppressor protein p53 by stimulating its ubiquitinylation and subsequent degradation. Ubiquitinylation is a multistep process involving a ubiquitin-activating enzyme, one of many distinct ubiquitin-conjugating enzymes, and in certain cases, a ubiquitin ligase. In human papillomavirus-infected cells, E6 and the E6-associated protein are thought to act as a ubiquitin-protein ligase in the ubiquitinylation of p53. Here we describe the cloning of a human ubiquitin-conjugating enzyme that specifically ubiquitinylates E6-associated protein. Furthermore, we define the biochemical pathway of p53 ubiquitinylation and demonstrate that in vivo inhibition of various components in the pathway leads to an inhibition of E6-stimulated p53 degradation.
PMCID:42146
PMID: 7724550
ISSN: 0027-8424
CID: 539972

Differential expression and cell cycle regulation of the cyclin-dependent kinase 4 inhibitor p16Ink4

Tam SW; Shay JW; Pagano M
p16Ink4 (inhibitor of cyclin-dependent kinase 4) is a cell cycle regulator that specifically binds to and inhibits Cdk4. Recently, the human mts1 (multiple tumor suppressor 1) gene, deleted or mutated in various primary tumors and in a large number of transformed cell lines, was found to be identical to ink4. In this study we have surveyed by immunoblotting the protein levels of p16Ink4 in normal and transformed human cells. We determined that p16Ink4 was differentially expressed in diploid cells derived from different tissues, in contrast to another cell cycle inhibitor, p21Waf1, which is ubiquitously expressed. In some tumor cell lines p16Ink4 protein was not detected, presumably because of a homozygous deletion of its gene. By contrast, it was found to be overexpressed in other cell lines when compared to levels in their normal counterparts. Interestingly, high levels of p16Ink4 protein correlated with functional inactivation of the retinoblastoma gene product. We also found that p16Ink4 protein expression varies during the cell cycle peaking during S phase. These results show a functional relationship between p16Ink4 and the retinoblastoma gene product and indicate that p16Ink4 is required for Cdk4 inhibition only at the G1-S transition at the time when Cdk4 kinase activity is no longer necessary
PMID: 7954407
ISSN: 0008-5472
CID: 21101

Differential expression and regulation of Cyclin D1 protein in normal and tumor human cells: association with Cdk4 is required for Cyclin D1 function in G1 progression

Tam SW; Theodoras AM; Shay JW; Draetta GF; Pagano M
In this study we have surveyed by immunoblotting the protein levels of Cyclin D1, D2, D3 and their catalytic partners, Cdk4 and Cdk6 in normal and transformed human cells. We found that all these proteins were differentially expressed in diploid cells derived from different tissues, in contrast to Cyclin E, Cyclin A and Cdk2 which are ubiquitously expressed. D-type Cyclins were never dramatically overexpressed and often very poorly expressed in tumor cell lines when compared to the levels in their normal counterparts. In contrast, Cdk4 was expressed at high levels in several tumor cell lines and Cdk6 was ectopically expressed in two sarcoma lines, suggesting a possible involvement of these two Cdks in oncogenesis. Interestingly, low levels of Cyclin D1 and D3 proteins always correlated with functional inactivation of the retinoblastoma gene product (pRb). In cells displaying active pRb, Cyclin D1 was found associated with Cdk4 regardless of whether the p53 gene was wild-type or mutant. Microinjection during G1 of Cyclin D1 anti-sense cDNA or anti-Cyclin D1 antibody in these cells arrested the cell cycle in G1. In cells lacking pRb function, Cyclin D1 was dissociated from Cdk4. Microinjection during G1 of Cyclin D1 antisense cDNA or anti-Cyclin D1 antibody in these cells did not affect G1 progression. These results show that (i) in the absence of pRb, Cyclin D1 is expressed at low levels, is dissociated from Cdk4 and becomes dispensable in G1; (ii) Cyclin D1 needs to be associated with its catalytic subunit, Cdk4, to function as a positive regulator of G1 progression
PMID: 8058330
ISSN: 0950-9232
CID: 21102

Cyclin D1-mediated inhibition of repair and replicative DNA synthesis in human fibroblasts

Pagano M; Theodoras AM; Tam SW; Draetta GF
Cyclin D1 is a key regulator of the G1 phase of the cell cycle. Inhibition of cyclin D1 function results in cell cycle arrest, whereas unregulated expression of the protein accelerates G1. Cyclin D1 is localized to the nucleus during G1. We found that during repair DNA synthesis, subsequent to UV-induced DNA damage, G1 cells readily lost their cyclin D1 while the proliferating cell nuclear antigen (PCNA) tightly associated with nuclear structures. Microinjection of cyclin D1 antisense accelerated DNA repair, whereas overexpression of cyclin D1 prevented DNA repair and the relocation of PCNA after DNA damage. Coexpression of cyclin D1 with its primary catalytic subunit, Cdk4, or with Cdk2, also prevented repair. In contrast, coexpression of PCNA, which is also a cyclin D1-associated protein, restored the ability of cells to repair their DNA. Acute overexpression of cyclin D1 in fibroblasts prevented them from entering S phase. Again, these effects were abolished by coexpression of cyclin D1 together with PCNA, but not with Cdk4 or Cdk2. Altogether, these results indicate that down-regulation of cyclin D1 is necessary for PCNA relocation and repair DNA synthesis as well as for the start of DNA replication. Cyclin D1 appears to be an essential component of a G1-checkpoint
PMID: 7958844
ISSN: 0890-9369
CID: 21103

Cyclin D1 protein oscillates and is essential for cell cycle progression in human tumour cell lines

Lukas, J; Pagano, M; Staskova, Z; Draetta, G; Bartek, J
Among the key cell cycle regulators, cyclin D1 has been implicated most strongly in oncogenesis. This G1 cyclin is a putative proto-oncogene whose clonal rearrangement and/or amplification and mRNA overexpression occurs in several types of human neoplasias. We have now raised a series of monoclonal antibodies to human cyclin D1 and analysed its regulation at the protein level in 40 human tumour cell lines. We found that 12 cell lines displayed low or undetectable cyclin D1 protein level, while the remaining lines accumulated the protein to a level comparable to, or moderately higher than, that of four normal diploid non-immortalized cell types. The cell cycle-dependent oscillation and subcellular localization of cyclin D1 were similar in both tumour and normal cells. The protein localized to the nucleus of G1 cells, and it was reduced to immunocytochemically undetectable level in DNA-replicating cells. At the functional level, microinjection and electroporation of anti-D1 antibodies revealed that in most tumour cell lines studied, including those with amplification at the cyclin D1 locus, this cyclin is essential for cell cycle progression in G1. Some tumours, however, seem to have evolved mechanism(s) that enable them to bypass the requirement for functional cyclin D1.
PMID: 8108113
ISSN: 0950-9232
CID: 539982