Timothy K. MacLachlan

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 G1 or G2 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.

BRCA1 Directs a Selective p53-Dependent Transcriptional Response towards Growth Arrest and DNA Repair Targets

ABSTRACT The pathway leading to BRCA1-dependent tumor suppression is not yet clear but appears to involve activities in DNA repair as well as gene transcription. Moreover, it has been shown that BRCA1 can regulate p53-dependent transcription. Because BRCA1 overexpression stabilizes wild-type p53 but does not lead to apoptosis of most cell lines, we investigated the selectivity of BRCA1 for p53-dependent target gene activation. We find that BRCA1-stabilized p53 regulates transcription of DNA repair and growth arrest genes while p53 stabilized by DNA-damaging agents induces a wide array of genes, including those involved in apoptosis. This differential expression profile was reflected in the treatment outcome—apoptosis following DNA damage and growth arrest after expression of BRCA1. Depletion of BRCA1 in wild-type-p53-expressing cells abolished the induction of such repair genes as p53R2, while the expression of PIG3, an apoptosis-inducing gene, was still induced. BRCA1 also conferred diminished cell death in a p53-dependent manner in response to adriamycin compared to that conferred by controls. These results suggest that BRCA1 selectively coactivates the p53 transcription factor towards genes that direct DNA repair and cell cycle arrest but not towards those that direct apoptosis.

Apoptotic threshold is lowered by p53 transactivation of caspase-6

Little is known about how a cells apoptotic threshold is controlled after exposure to chemotherapy, although the p53 tumor suppressor has been implicated. We identified executioner caspase-6 as a transcriptional target of p53. The mechanism involves DNA binding by p53 to the third intron of the caspase-6 gene and transactivation. A p53-dependent increase in procaspase-6 protein level allows for an increase in caspase-6 activity and caspase-6-specific Lamin A cleavage in response to Adriamycin exposure. Specific inhibition of caspase-6 blocks cell death in a manner that correlates with caspase-6 mRNA induction by p53 and enhances long-term survival in response to a p53-mediated apoptotic signal. Caspase-6 is an executioner caspase found directly regulated by p53, and the most downstream component of the death pathway controlled by p53. The induction of caspase-6 expression lowers the cell death threshold in response to apoptotic signals that activate caspase-6. Our results provide a potential mechanism of lowering the death threshold, which could be important for chemosensitization.

BRCA1 Transcriptionally Regulates Damaged DNA Binding Protein (DDB2) In the DNA Repair Response Following UV-Irradiation

The p53 and BRCA1 tumor suppressors are involved in repair processes and may cooperate to transactivate certain genes, including p21WAF1/CIP1 and GADD45. We find that the Xeroderma Pigmentosum Complementation group E (XPE) mutated Damaged-DNA binding protein p48 (DDB2) is upregulated by BRCA1 in a p53-dependent manner following UVC, Adriamycin, or Cisplatin exposure. BRCA1 enhances p53 binding to the DDB2 promoter in vivo as well as p53-dependent transactivation of DDB2 promoter-reporter constructs through a classical p53 DNA responsive element. Antisense abrogation of BRCA1 expression abrogates upregulation of DDB2 after UVC or cisplatin exposure. Using a host cell reactivation assay, DNA repair activity is more significantly restored by introduction of BRCA1 into wt as compared to DDB2-deficient cells. Furthermore disappearance of the photoproducts cyclobutane pyrimidine dimer (CPD) and 6-4 photoproduct (6-4PP) was delayed by antisense abrogation of BRCA1 expression in UV-exposed human cells. Thus the DNA repair function of BRCA1 may be attributed in part to p53-dependent transcriptional induction of DDB2. Loss of BRCA1-dependent DDB2 repair function may contribute to cancer susceptibility and cellular sensitivity to DNA damage.

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.

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 BRCA1promoter-driven luciferase activity even in null p21 cells deficient in p53-dependent G1 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.

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 CDK9s 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. J. Cell. Biochem. 71:467–478, 1998.

Insulin-Like Growth Factor Factor Binding Protein-2 is a Novel Mediator of p53 Inhibition of Insulin-Like Growth Factor Signaling

The p53 tumor suppressor induces cellular growth arrest and apoptosis in response to DNA damage by transcriptionally activating or repressing target genes and also through protein-protein interactions and direct mitochondrial activities. In 1995, insulin-like growth factor binding protein (IGFBP)-3 was identified as one of the genes transcriptionally activated by p53. IGFBP-3 is one of six closely related IGFBP’s, with additional IGFBP-related proteins belonging to the IGFBP superfamily. Here we show that IGFBP-2 is also a p53 target. Like IGFBP-3, IGFBP-2 secretion is reduced when p53+/+ lung cancer cells are transfected with human papillomavirus E6, which targets p53 for degradation. IGFBP-2 mRNA is induced by irradiation in vivo in a p53-dependent manner. p53 protein binds IGFBP-2 intronic sequences in an electrophoretic mobility shift assay, and activates transcription in a luciferase assay. Loss of IGFBP-2 inhibits the ability of p53 to inhibit the activation of extracellular signal-regulated kinase (ERK)1 by IGF-I. Thus, p53 effects on the IGF axis are more complex than previously appreciated, and overall transform the axis from IGF-mediated mitogenesis to growth inhibition and apoptosis. This has significant implications for how growth hormone and IGF-I can induce growth without also inducing cancer.

Structure-based design of p18INK4c proteins with increased thermodynamic stability and cell cycle inhibitory activity.

p18INK4c is a member of the INK4 family of proteins that regulate the G1 to S cell cycle transition by binding to and inhibiting the pRb kinase activity of cyclin-dependent kinases 4 and 6. The p16INK4a member of the INK4 protein family is altered in a variety of cancers and structure-function studies of the INK4 proteins reveal that the vast majority of missense tumor-derived p16INK4a mutations reduce protein thermodynamic stability. Based on this observation, we used p18INK4c as a model to test the proposal that INK4 proteins with increased stability might have enhanced cell cycle inhibitory activity. Structure-based mutagenesis was used to prepare p18INK4c mutant proteins with a predicted increase in stability. Using this approach, we report the generation of three mutant p18INK4C proteins, F71N, F82Q, and F92N, with increased stability toward thermal denaturation of which the F71N mutant also showed an increased stability to chemical denaturation. The x-ray crystal structures of the F71N, F82Q, and F92N p18INK4C mutant proteins were determined to reveal the structural basis for their increased stability properties. Significantly, the F71N mutant also showed enhanced CDK6 interaction and cell cycle inhibitory activity in vivo, as measured using co-immunoprecipitation and transient transfection assays, respectively. These studies show that a structure-based approach to increase the thermodynamic stability of INK4 proteins can be exploited to prepare more biologically active molecules with potential applications for the development of molecules to treat p16INK4a-mediated cancers.

Insights into differential endostatin activity

commentary to Opposite Effects of Endostatin on Different Endothelial Cells Annette Schmidt, Klaus Addicks and Wilhelm Bloch