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Biomedical subjects

J W Harper

Publications and source records attributed to J W Harper.

At least 55 records · Page 3Linked to original sources
PubMed1997

Cyclin dependent kinase inhibitors.

Progression through the eukaryotic cell cycle is regulated by the activities of a family of cyclin dependent kinases (CDKs). These kinases are negatively regulated by phosphorylation and by the action of cyclin kinase inhibitors (CKIs). In mammalian cells, two classes of CKIs have been identified, the INK4 class and the CIP/KIP class. These CKIs are versatile negative regulators of CDK function and have potential roles in development, checkpoint control and tumour suppression. Analysis of CKI knockout indicates that although these inhibitors are not generally required for survival, the phenotypes observed span the gamut of what might be expected for loss of a cell cycle inhibitor. This chapter summarizes our current understanding of the roles of CKIs in growth control.

Animals↗
PubMed1996

SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box.

We have identified the yeast and human homologs of the SKP1 gene as a suppressor of cdc4 mutants and as a cyclin F-binding protein. Skp1p indirectly binds cyclin A/Cdk2 through Skp2p, and directly binds Skp2p, cyclin F, and Cdc4p through a novel structural motif called the F-box. SKP1 is required for ubiquitin-mediated proteolysis of Cin2p, Clb5p, and the Cdk inhibitor Sic1p, and provides a link between these molecules and the proteolysis machinery. A large number of proteins contain the F-box motif and are thereby implicated in the ubiquitin pathway. Different skp1 mutants arrest cells in either G1 or G2, suggesting a connection between regulation of proteolysis in different stages of the cycle.

Amino Acid Sequence↗
PubMed1996

Mammalian p50Cdc37 is a protein kinase-targeting subunit of Hsp90 that binds and stabilizes Cdk4.

CDC37, an essential gene in Saccharomyces cerevisiae, interacts genetically with multiple protein kinases and is required for production of Cdc28p/cyclin complexes through an unknown mechanism. We have identified mammalian p50Cdc37 as a protein kinase-targeting subunit of the molecular chaperone Hsp90. Previously, p50 was observed in complexes with pp60v-src and Raf-1, but its identity and function have remained elusive. In mouse fibroblasts, a primary target of Cdc37 is Cdk4. This kinase is activated by D-type cyclins and functions in passage through G1. In insect cells, Cdc37 is sufficient to target Hsp90 to Cdk4 and both in vitro and in vivo, Cdc37/Hsp90 associates preferentially with the fraction of Cdk4 not bound to D-type cyclins. Cdc37 is coexpressed with cyclin Dl in cells undergoing programmed proliferation in vivo, consistent with a positive role in cell cycle progression. Pharmacological inactivation of Cdc37/Hsp90 function decreases the half-life of newly synthesized Cdk4, indicating a role for Cdc37/Hsp90 in Cdk4 stabilization. This study suggests a general role for p50Cdc37 in signaling pathways dependent on intrinsically unstable protein kinases and reveals a previously unrecognized chaperone-dependent step in the production of Cdk4/cyclin D complexes.

Amino Acid Sequence↗
PubMed1996

Imprinting of the gene encoding a human cyclin-dependent kinase inhibitor, p57KIP2, on chromosome 11p15.

Parental origin-specific alterations of chromosome 11p15 in human cancer suggest the involvement of one or more maternally expressed imprinted genes involved in embryonal tumor suppression and the cancer-predisposing Beckwith-Wiedemann syndrome (BWS). The gene encoding cyclin-dependent kinase inhibitor p57KIP2, whose overexpression causes G1 phase arrest, was recently cloned and mapped to this band. We find that the p57KIP2 gene is imprinted, with preferential expression of the maternal allele. However, the imprint is not absolute, as the paternal allele is also expressed at low levels in most tissues, and at levels comparable to the maternal allele in fetal brain and some embryonal tumors. The biochemical function, chromosomal location, and imprinting of the p57KIP2 gene match the properties predicted for a tumor suppressor gene at 11p15.5. However, as the p57KIP2 gene is 500 kb centromeric to the gene encoding insulin-like growth factor 2, it is likely to be part of a large domain containing other imprinted genes. Thus, loss of heterozygosity or loss of imprinting might simultaneously affect several genes at this locus that together contribute to tumor and/or growth- suppressing functions that are disrupted in BWS and embryonal tumors.

Base Sequence↗
PubMed1996

Selective maternal-allele loss in human lung cancers of the maternally expressed p57KIP2 gene at 11p15.5.

Genomic imprinting at 11p15 is suggested to play a role in certain pediatric tumors such as Wilms' tumor, based on the findings of selective maternal loss of this chromosomal region. Although the allele loss at 11p15 is also frequent in a number of cancers of adults including lung, breast, and bladder cancers, possible involvement of genomic imprinting in these tumors has not been investigated extensively. p57KIP2, a newly described member of the p21 cyclin-dependent kinase (CDK) inhibitor family which is thought to negatively regulate the cell cycle at the G1 checkpoint, has been mapped to 11p15. In the present study, we searched for somatic p57KIP2 mutations in lung cancer, but failed to find such alterations. Interestingly, however, we found that the p57KIP2 gene is imprinted with maternal expression and that the maternal alleles had been selectively lost in 11 of 13 (85%) lung cancer cases carrying 11p15 deletions, this being a significant bias (p=0.01). These data provide the first evidence that genomic imprinting may play a role in the oncogenesis of not only rare pediatric tumors but also this common cancer of adults, suggesting that the imprinted p57KIP2 CDK inhibitor gene is a potential target for maternally biased 11p15 deletions.

Adult↗
PubMed1996

Temporal study of the activity of matrix metalloproteinases and their endogenous inhibitors during wound healing.

The restoration of functional connective tissue is a major goal of the wound healing process. This regenerative event requires the deposition and accumulation of collagenous and noncollagenous matrix molecules as well as the remodelling of extracellular matrix (ECM) by matrix metalloproteinases (MMPs). In this study, we have utilized substrate gel electrophoresis, radiometric enzyme assays, and Western blot analyses to determine the temporal pattern of appearance and activity of active and latent MMPs and their inhibitors during the entire healing process in a partial thickness wound model. Through the use of substrate gel electrophoresis, we studied the appearance of proteolytic bands whose molecular weight was consistent with their being members of the MMP family of enzymes. Proteolytic bands whose molecular weight is consistent with both the active and latent forms of MMP-2 (72 kDa, Type IV gelatinase) were detected in wound fluid of days 1-7 after wounding. The number of active MMP-2 species detectable in wound fluid was greatest during days 4-6 after wounding. The most prominent proteolytic band detected each day migrated with a molecular weight consistent with it being the latent form of MMP-9 (92 kDa, Type V pro-collagenase). In contrast to MMP-2, the active form of this enzyme was never detected. The presence of MMP-1 (interstitial collagenase) was detected by immunoblot in the wound fluid from days 1-6 post-injury. Using a radiometric enzyme assay for collagenase inhibitory activity we have also determined the time course of activity of endogenous matrix metalloproteinase inhibitors. We have correlated these data to the known cellular events occurring in the wound during this time period as well. This study establishes a prototypical pattern of MMP appearance in normal wound healing. It may also provide potential intervention sites for the therapeutic use of inhibitors of aberrant MMP activities which characterize chronic wounds.

Animals↗
PubMed1996

A question of balance: the role of cyclin-kinase inhibitors in development and tumorigenesis.

Cyclin-kinase inhibitors (CKIs) are versatile negative regulators of cell proliferation that function in developmental decisions, checkpoint control and tumour suppression. Phenotypic examination of mice lacking individual CKIs has begun to reveal the specialized roles that each of these proteins play in vivo. This review focuses on what has been learned about the role of CKIs in development and cancer through the generation of knockout animals. The authors discuss whether differences in knockout phenotypes between CKIs reflect differential use of these inhibitors by the organism or a fundamental difference between the inhibitors, and suggest a balance hypothesis to explain the different effects observed.

Journal Article↗
PubMed1996

Cdk inhibitors in development and cancer.

Activation of cyclin-dependent kinases is the primary control point of cell proliferation. Recent advances in the understanding of cell-cycle regulation have uncovered two families of proteins that bind and inhibit these kinases. These proteins are likely mediators of development and differentiation, and may provide molecular connections between the pathways of development and tumorigenesis.

Animals↗
PubMed1995

In vivo gene therapy with p53 or p21 adenovirus for prostate cancer.

We introduced the gene for wild-type human p53 or p21, a critical downstream mediator of p53-induced growth suppression, into a p53-deficient mouse prostate cancer cell line using a recombinant adenoviral vector (Ad5CMV-p53 or Ad5CMV-p21). Elevated levels of endogenous mouse p21 mRNA provided evidence for the functional activity of virally transduced p53. Functional activity of viral-transduced p21 was demonstrated through immunoprecipitation of cellular protein extracts, which showed that the viral-transduced p21 associates with cyclin-dependent kinase 2 and was sufficient to down-regulate the activity of the cyclin-dependent kinase by approximately 65%. In vitro growth assays revealed significantly higher growth suppression after Ad5CMV-p21 infection compared to Ad5CMV-p53. In vivo studies in syngeneic male mice with established s.c. prostate tumors demonstrated that the rate of growth and final tumor volume were reduced to a much greater extent in mice that received intratumor injection of Ad5CMV-p21 compared to Ad5CMV-p53. In addition, the survival of host animals bearing tumors that were infected with Ad5CMV-p21, but not Ad5CMV-p53, was significantly extended. These data suggest that Ad5CMV-p21 may be effective as a therapeutic agent for prostate cancer.

Adenoviridae↗
PubMed1995

Mice lacking p21CIP1/WAF1 undergo normal development, but are defective in G1 checkpoint control.

p21CIP1/WAF1 is a CDK inhibitor regulated by the tumor suppressor p53 and is hypothesized to mediate G1 arrest. p53 has been suggested to derive anti-oncogenic properties from this relationship. To test these notions, we created mice lacking p21CIP1/WAF1. They develop normally and (unlike p53-/- mice) have not developed spontaneous malignancies during 7 months of observation. Nonetheless, p21-/- embryonic fibroblasts are significantly deficient in their ability to arrest in G1 in response to DNA damage and nucleotide pool perturbation. p21-/- cells also exhibit a significant growth alteration in vitro, achieving a saturation density as high as that observed in p53-/- cells. In contrast, other aspects of p53 function, such as thymocytic apoptosis and the mitotic spindle checkpoint, appear normal. These results establish the role of p21CIP1/WAF1 in the G1 checkpoint, but suggest that the anti-apoptotic and the anti-oncogenic effects of p53 are more complex.

Animals↗
PubMed1995

p57KIP2, a structurally distinct member of the p21CIP1 Cdk inhibitor family, is a candidate tumor suppressor gene.

Cyclin-dependent kinases (Cdks) are positive regulators of cell proliferation, whereas Cdk inhibitors (CKIs) inhibit proliferation. We describe a new CKI, p57KIP2, which is related to p21CIP1 and p27KIP1. p57KIP2 is a potent, tight-binding inhibitor of several G1 cyclin/Cdk complexes, and its binding is cyclin dependent. Unlike CIP1, KIP2 is not regulated by p53. Overexpression of p57KIP2 arrests cells in G1. p57KIP2 proteins have a complex structure. Mouse p57KIP2 consists of four structurally distinct domains: an amino-terminal Cdk inhibitory domain, a proline-rich domain, an acidic-repeat region, and a carboxy-terminal domain conserved with p27KIP1. Human p57KIP2 appears to have conserved the amino- and carboxy-terminal domains but has replaced the internal regions with sequences containing proline-alanine repeats. In situ hybridization during mouse embryogenesis revealed that KIP2 mRNA displays a striking pattern of expression during development, showing high level expression in skeletal muscle, brain, heart, lungs, and eye. Most of the KIP2-expressing cells are terminally differentiated, suggesting that p57KIP2 is involved in decisions to exit the cell cycle during development and differentiation. Human KIP2 is located at 11p15.5, a region implicated in both sporadic cancers and Beckwith-Wiedemann syndrome, a familial cancer syndrome, marking it as a candidate tumor suppressor. The discovery of a new member of the p21CIP1 inhibitor family with novel structural features and expression patterns suggests a complex role for these proteins in cell cycle control and development.

Amino Acid Sequence↗
PubMed1995

p53-independent expression of p21Cip1 in muscle and other terminally differentiating cells.

Terminal differentiation is coupled to withdrawal from the cell cycle. The cyclin-dependent kinase inhibitor (CKI) p21Cip1 is transcriptionally regulated by p53 and can induce growth arrest. CKIs are therefore potential mediators of developmental control of cell proliferation. The expression pattern of mouse p21 correlated with terminal differentiation of multiple cell lineages including skeletal muscle, cartilage, skin, and nasal epithelium in a p53-independent manner. Although the muscle-specific transcription factor MyoD is sufficient to activate p21 expression in 10T1/2 cells, p21 was expressed in myogenic cells of mice lacking the genes encoding MyoD and myogenin, demonstrating that p21 expression does not require these transcription factors. The p21 protein may function during development as an inducible growth inhibitor that contributes to cell cycle exit and differentiation.

Animals↗
PubMed1995

Inhibition of cyclin-dependent kinases by p21.

p21Cip1 is a cyclin-dependent kinase (Cdk) inhibitor that is transcriptionally activated by p53 in response to DNA damage. We have explored the interaction of p21 with the currently known Cdks. p21 effectively inhibits Cdk2, Cdk3, Cdk4, and Cdk6 kinases (Ki 0.5-15 nM) but is much less effective toward Cdc2/cyclin B (Ki approximately 400 nM) and Cdk5/p35 (Ki > 2 microM), and does not associate with Cdk7/cyclin H. Overexpression of P21 arrests cells in G1. Thus, p21 is not a universal inhibitor of Cdks but displays selectivity for G1/S Cdk/cyclin complexes. Association of p21 with Cdks is greatly enhanced by cyclin binding. This property is shared by the structurally related inhibitor p27, suggesting a common biochemical mechanism for inhibition. With respect to Cdk2 and Cdk4 complexes, p27 shares the inhibitory potency of p21 but has slightly different kinase specificities. In normal diploid fibroblasts, the vast majority of active Cdk2 is associated with p21, but this active kinase can be fully inhibited by addition of exogenous p21. Reconstruction experiments using purified components indicate that multiple molecules of p21 can associate with Cdk/cyclin complexes and inactive complexes contain more than one molecule of p21. Together, these data suggest a model whereby p21 functions as an inhibitory buffer whose levels determine the threshold kinase activity required for cell cycle progression.

Binding, Competitive↗
PubMed1995

Inhibition of HIV-1 by a double transdominant fusion gene.

A double transdominant fusion gene (trev) designed to inhibit two essential HIV functions simultaneously was constructed by linking tat and rev transdominant mutants. Trev independently inhibited both Tat and Rev functions, localized within the nucleus and cells transfected with trev showed a stable inhibition of HIV-1-mediated cytopathicity. A retroviral vector of trev was made and shown also to confer protection from HIV cytopathic effects. Simultaneous inhibition of two essential viral genes presents significant advantages for potential gene therapy treatment of HIV infection over conventional single effect molecules.

Amino Acid Sequence↗
PubMed1994

Deletion of p16 and p15 genes in brain tumors.

We have used molecular genetic methods to examine the status of cell cycle-inhibitory genes in human brain tumors. We found that p16 and a neighboring gene, p15, were often homozygously deleted in glioblastoma multiformes but not in medulloblastomas or ependymomas. The deletions occurred in both primary tumors and their derived xenografts, but no intragenic mutations in either of the two genes were found. The p15 gene was expressed in a more widespread pattern in normal tissues than p16, but the products of both genes had similar capacities to bind to cyclin D-dependent kinases 4 and 6. These data suggest that the target of deletion in glioblastoma multiforme includes both p15 and p16 genes. The reason that homozygous deletions, rather than intragenic mutations, are so common in these tumors may be that deletion is a more efficient mechanism for simultaneous inactivation of both genes.

Animals↗
PubMed1994

Cdk-interacting protein 1 directly binds with proliferating cell nuclear antigen and inhibits DNA replication catalyzed by the DNA polymerase delta holoenzyme.

Cdk-interacting protein 1 (Cip1) is a p53-regulated 21-kDa protein that inhibits several members of the cyclin-dependent kinase (CDK) family. It was initially observed in complexes containing CDK4, cyclin D, and proliferating cell nuclear antigen (PCNA). PCNA, in conjunction with activator 1, acts as a processivity factor for eukaryotic DNA polymerase (pol) delta, and these three proteins constitute the pol delta holoenzyme. In this report, we demonstrate that Cip1 can also directly inhibit DNA synthesis in vitro by binding to PCNA. Cip1 efficiently inhibits simian virus 40 replication dependent upon pol alpha, activator 1, PCNA, and pol delta, and this inhibition can be overcome by additional PCNA. Simian virus 40 DNA replication, catalyzed solely by high levels of pol alpha-primase complex, is unaffected by Cip1. Using the surface plasmon resonance technique, a direct physical interaction of PCNA and Cip1 was detected. We have observed that Cip1 efficiently inhibits synthesis of long (7.2 kb) but not short (10 nt) templates, suggesting that its association with PCNA is likely to impair the processive movement of pol delta during DNA chain elongation, as opposed to blocking assembly of the pol delta holoenzyme. The implications of the Cip1-PCNA interaction with respect to regulation of DNA synthesis, cell cycle checkpoint control, and DNA repair are discussed.

CDC2-CDC28 Kinases↗
PubMed1994

p53-dependent inhibition of cyclin-dependent kinase activities in human fibroblasts during radiation-induced G1 arrest.

gamma-Irradiation of human diploid fibroblasts in the G1 interval caused arrest of the cell cycle prior to S phase. This cell cycle block was correlated with a lack of activation of both cyclin E-Cyclin-dependent kinase 2 (Cdk2) and cyclin A-Cdk2 kinases and depended on wild-type p53. Although the accumulation of cyclin A was strongly inhibited in gamma-irradiated cells, cyclin E accumulated and bound Cdk2 at normal levels but remained in an inactive state. We found that both whole-cell lysates and inactive cyclin E-Cdk2 complexes prepared from irradiated cells contained an activity capable of inactivating cyclin E-Cdk2 complexes. The protein responsible for this activity was shown to be p21CIP1/WAF1, recently described as a p53-inducible Cdk inhibitor. Our data suggest a model in which ionizing radiation confers G1 arrest via the p53-mediated induction of a Cdk inhibitor protein.

Base Sequence↗