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Biomedical subjects

T K MacLachlan

Publications and source records attributed to T K MacLachlan.

14 recordsLinked to original sources
PubMed2000

Repression of BRCA1 through a feedback loop involving p53.

The BRCA1 and p53 tumor suppressors have been shown to interact and cooperate to activate transcription of p53-responsive genes. In this study, we show that BRCA1 is initially up-regulated, followed by a reduction to below basal levels in response to treatment with the DNA-damaging agents adriamycin and mitomycin C, and that the reduction of BRCA1 expression is dependent on the presence of wild-type p53. Elimination of p53 by expression of human papilloma virus E6 resulted in an inability to down-regulate BRCA1 in response to adriamycin. Ectopic expression of p53 resulted in a rapid decrease in BRCA1 protein and RNA levels and BRCA1 promoter-driven luciferase activity even in null p21 cells deficient in p53-dependent G(1) arrest. ATM(-)(/-) lymphoblastoid cells were deficient in their ability to reduce BRCA1 protein in response to DNA damage, whereas the wild-type counterparts reduced BRCA1 protein levels after exposure to adriamycin. These results, in conjunction with others, suggest a loop wherein BRCA1 initially participates in accumulation of p53 protein, whereas later p53 acts to reduce BRCA1 expression.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2000

BRCA1 effects on the cell cycle and the DNA damage response are linked to altered gene expression.

The breast and ovarian cancer susceptibility gene product BRCA1 has been reported to be expressed in a cell cycle-dependent manner; possess transcriptional activity; associate with several proteins, including the p53 tumor suppressor; and play an integral role in certain types of DNA repair. We show here that ectopic expression of BRCA1 using an adenovirus vector (Ad-BRCA1) leads to dephosphorylation of the retinoblastoma protein accompanied by a decrease in cyclin-dependent kinase activity. Flow cytometric analysis on Ad-BRCA1-infected cells revealed a G(1) or G(2) phase accumulation. High density cDNA array screening of colon, lung, and breast cancer cells identified several genes affected by BRCA1 expression in a p53-independent manner, including DNA damage response genes and genes involved in cell cycle control. Notable changes included induction of the GADD45 and GADD153 genes and a reduction in cyclin B1 expression. Therefore, BRCA1 has the potential to modulate the expression of genes and function of proteins involved in cell cycle control and DNA damage response pathways.

Adenoviridae↗
PubMed1999

BRCA1 signals ARF-dependent stabilization and coactivation of p53.

The hereditary breast and ovarian tumor suppressor BRCA1 can activate p53-dependent gene expression. We show here that BRCA1 increases p53 protein levels through a post-transcriptional mechanism. BRCA1-stabilized p53 has increased sequence-specific DNA-binding and transcriptional activity. BRCA1 does not stabilize p53 in p14ARF-deficient cells. A deletion mutant of BRCA1 which inhibits p53-dependent transcription confers resistance to topoisomerase II-targeted chemotherapy. Our results suggest that BRCA1 may trigger the p53 pathway through two potentially separate mechanisms: accumulation of p53 through a direct or indirect induction of p14ARF as well as direct transcriptional coactivation of p53. BRCA1 may also enhance chemosensitivity and repair of DNA damage through binding to and coactivation of p53.

ADP-Ribosylation Factors↗
PubMed1999

Comparative gene expression profiling in response to p53 in a human lung cancer cell line.

The p53 tumor suppressor gene functions through the p53-mediated transcriptional activation and regulation of critical downstream target genes. The mechanism behind such regulation, however, remains unclear. In this study, we compared the expression of 30 genes that are involved in cell cycle checkpoint control and/or apoptosis in a human lung cancer cell line, which contains endogenous wild-type p53, in response to ectopic p53 expression. Of the 30 genes studied, 22 genes have shown an increase in expression. The increase in gene expression of 2 genes-Gadd45 and PIG2-was more than 10-fold. These results suggest that the genes with the highest expression level in a p53-dependent pathway may play a dominant role in determining the pathway that the cell follows: cell cycle arrest or apoptosis. Our screen illustrates the development of a simple and inexpensive p53-specific cDNA array to begin to analyze downstream events in the p53 pathway under physiological, pathological, and stress-induced states.

Apoptosis↗
PubMed1998

TRAF2 expression in differentiated muscle.

Recent data involving traf2 knockout mice have suggested a necessity of the protein in viability of skeletal muscle tissue. traf2 -/- mice are born with decreased muscle mass that is hypothesized to be due to the increased circulating tumor necrosis factor in these mice. We show that TRAF2 protein is present at high levels in terminally differentiated skeletal muscle in the developing mouse. In vitro differentiation of mouse myoblasts displays a dramatic increase in TRAF2 protein levels. Although basal NF-kappaB activity decreases during myogenesis, TNF-induced NF-kappaB activity is 10 times greater in myotubes compared with myoblasts, presumably because of the stockpiling of TRAF2 protein in these cells. This may represent a strong anti-apoptotic TRAF2-mediated response specifically tailored to myotubes. These data help explain why muscle integrity is at risk in traf2 -/- mice.

Animals↗
PubMed1998

Binding of CDK9 to TRAF2.

CDK9 has been recently shown to have increased kinase activity in differentiated cells in culture and a differentiated tissue-specific expression in the developing mouse. In order to identify factors that contribute to CDK9's differentiation-specific function, we screened a mouse embryonic library in the yeast two-hybrid system and found a tumor necrosis factor signal transducer, TRAF2, to be an interacting protein. CDK9 interacts with a conserved domain in the TRAF-C region of TRAF2, a motif that is known to bind other kinases involved in TRAF-mediated signaling. Endogenous interaction between the two proteins appears to be specific to differentiated tissue. TRAF2-mediated signaling may incorporate additional kinases to signal cell survival in myotubes, a cell type that is severely affected in TRAF2 knockout mice.

Animals↗
PubMed1998

Cloning of murine CDK9/PITALRE and its tissue-specific expression in development.

The cdc2-family of serine/threonine kinases and their binding partners recently were implicated in developmental roles. We previously cloned a cdc2-related kinase, cdk9/PITALRE, that is able to phosphorylate the retinoblastoma protein in vitro. We describe here the cloning and the characterization of the mouse homolog of cdk9/PITALRE. The murine cDNA is 98% identical with humans and is expressed at high levels in brain and kidney tissues. The kinase activity and protein expression of cdk9/PITALRE were highest in terminally differentiated tissues such as the muscle and brain. In situ immunohistology and immunofluorescence detected cdk9/PITALRE protein not only within terminally differentiated cells such as muscle and neuronal cells, but also in proliferating cells. C2C12 and P19 cells induced to differentiate along muscle and neural lineages peaked in cdk9/PITALRE kinase activity at the end of differentiation. These results suggest that, among other roles, cdk9/PITALRE plays a role not unlike cdk5 in the differentiation of certain cell types.

Amino Acid Sequence↗
PubMed1997

A unique domain of pRb2/p130 acts as an inhibitor of Cdk2 kinase activity.

The Cdk2 kinase has long been known to be involved in the progression of mammalian cells past the G1 phase restriction point and through DNA replication in the cell cycle. The Rb family of proteins, consisting of pRb, p107, and pRb2/p130, has also been shown to monitor progression of G1 phase, mostly through their interaction with E2F family members. p107 is able to inhibit Cdk2 kinase activity through this interaction via a p21-related domain present in the C terminus of the protein. We show here that pRb2/p130 also possesses this activity, but through a separate domain. Moreover, we correlate the increased expression of pRb2/p130 during various cellular processes with the decreased kinase activity of Cdk2. We hypothesize that pRb2/p130 may act not only to bind and modify E2F activity, but also to inhibit Cdk2 kinase activity in concert with p21 in a manner different from p107.

Animals↗
PubMed1997

Post-proliferative cyclin E-associated kinase activity in differentiated osteoblasts: inhibition by proliferating osteoblasts and osteosarcoma cells.

Spontaneous differentiation of normal diploid osteoblasts in culture is accompanied by increased cyclin E associated kinase activity on (1) the retinoblastoma susceptibility protein pRB, (2) the p107 RB related protein, and (3) two endogenous cyclin E-associated substrates of 78 and 105 kD. Activity of the differentiation-related cyclin E complexes (diff.ECx) is not recovered in cdc2 or cdk2 immunoprecipitates. Phosphorylation of both the 105 kD endogenous substrate and the p107 exogenous substrate is sensitive to inhibitory activity (diff.ECx-i) present in proliferating osteoblasts. This inhibitory activity is readily recruited by the cyclin E complexes of differentiated osteoblasts but is not found in cyclin E immunoprecipitates of the proliferating cells themselves. Strong inhibitory activity on diff.ECx kinase activity is excerted by proliferating ROS 17/2.8 osteosarcoma cells. However, unlike the normal diploid cells, the diff.ECx-i activity of proliferating ROS 17/2.8 cells is recovered by cyclin E immunoprecipitation. The cyclin-dependent kinase inhibitor p21CIP1/WAF1 inhibits diff.ECx kinase activity. Thus, our results suggest the existence of a unique regulatory system, possibly involving p21CIP1/WAF1, in which inhibitory activity residing in proliferating cells is preferentially targeted towards differentiation-related cyclin E-associated kinase activity.

Animals↗
PubMed1995

The cdc-2-related kinase, PISSLRE, is essential for cell growth and acts in G2 phase of the cell cycle.

Mammalian cell cycle progression is regulated by several protein kinases that are activated by cyclically expressed proteins called cyclins. These cyclin-dependent kinases, the prototype of which is the cdc2 mitosis-promoting kinase, are known to phosphorylate substrates the modified status of which is critical for the cell to progress into sequential phases of the cycle. Recently, a new cdc2-related protein kinase has been discovered. PISSLRE, named with respect to its homology to the cdc2 PSTAIRE amino acid domain. Here we report that by using both antisense and dominant-negative mutant constructs of PISSLRE when overexpressed in U2OS cells, a growth suppression is found. Furthermore, the dominant negative forms of PISSLRE halt cell cycle progression in G2-M. Therefore, PISSLRE is essential for cellular proliferation, and its effect is exerted in G2-M. This describes the first evidence since cdc2 of a cdc2-related kinase acting through G2-M.

Amino Acid Sequence↗
PubMed1995

Chromosomal mapping of members of the cdc2 family of protein kinases, cdk3, cdk6, PISSLRE, and PITALRE, and a cdk inhibitor, p27Kip1, to regions involved in human cancer.

Orderly progression through the cell cycle requires sequential activation and inactivation of cyclin-dependent kinases (cdks). This is achieved in part through the association of cdks with positive regulators called cyclins and inactivation of cyclin-cdk complexes by a rapidly growing number of cyclin-cdk inhibitors. Recently, the role of cell cycle control proteins both as primary effectors and as mediators of tumorigenesis has become a subject of increased interest. Here we report the chromosomal mapping of two cdks, cdk3 and cdk6, two putative cdks, PISSLRE and PITALRE, and one cyclin-dependent kinase inhibitor, p27, to chromosomal regions which may be altered in human tumors and examine their possible involvement in some of these malignancies. In particular, two of the kinases, cdk3 and PISSLRE and PITALRE, the cdc2-related kinases recently cloned by us, map to regions previously shown to exhibit loss of heterozygosity in breast and other tumors.

Base Sequence↗
PubMed1995

Cyclins, cyclin-dependent kinases and cdk inhibitors: implications in cell cycle control and cancer.

A significant portion of cell scientific literature published is dedicated to describing the cloning, the link to cancer, or the characterization of proteins involved in the progression of the cell cycle. With this abundance of information, the cascading pathways of molecular events that occur in the cell cycle are proving to be exceedingly complicated. Originally, the sole regulator of the fission yeast cells division cycle, cdc2, was thought to also regulate mammalian cell cycles in the same manner. However, mammalian cdc2 has now been joined by seven well-characterized relatives acting at distinct points in the cell cycle. These kinases are activated by larger proteins called cyclins, named with respect to their cyclical expression and degradation. Therefore, the catalytic subunits of these complexes are named cyclin-dependent kinases (cdks). In the event that the cell must stop normal cycling behavior, a number of cdk inhibitors, which have only begun to be characterized, function in inhibiting the kinase ability of cdks, among other nonproliferative acts. The external environment manipulates cellular proliferation and differentiation by stimulating or inhibiting certain signal transduction pathways. However, each component of the cell cycle machinery, as they are the final executors in cell division, has the potential to elicit or to contribute to a neoplastic phenotype. This review focuses on the characterization of each member of the cell cycle protein family and also addresses the potential role each plays in cancer.

Animals↗