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Jeffrey D Parvin - One of the best experts on this subject based on the ideXlab platform.

  • a multiplexed homology directed dna repair assay reveals the impact of 1 700 BRCA1 variants on Protein function
    bioRxiv, 2018
    Co-Authors: Lea M Starita, Muhtadi M Islam, Tapahsama Banerjee, Aleksandra I Adamovich, Justin Gullingsrud, Stanley Fields, Jay Shendure, Jeffrey D Parvin
    Abstract:

    Loss-of-function mutations in BRCA1 confer a predisposition to breast and ovarian cancer. Genetic testing for mutations in the BRCA1 gene frequently reveals a missense variant for which the impact on the molecular function of the BRCA1 Protein is unknown. Functional BRCA1 is required for homology directed repair (HDR) of double-strand DNA breaks, a key activity for maintaining genome integrity and tumor suppression. Here we describe a multiplex HDR reporter assay to simultaneously measure the effect of hundreds of variants of BRCA1 on its role in DNA repair. Using this assay, we measured the effects of ~1,700 amino acid substitutions in the first 302 residues of BRCA1. Benchmarking these results against variants with known effects, we demonstrate accurate discrimination of loss-of-function versus benign variants. We anticipate that this assay can be used to functionally characterize BRCA1 missense variants at scale, even before the variants are observed in results from genetic testing.

  • centrosomal microtubule nucleation activity is inhibited by BRCA1 dependent ubiquitination
    Molecular and Cellular Biology, 2005
    Co-Authors: Satish Sankaran, Aaron C. Groen, Lea M Starita, Jeffrey D Parvin
    Abstract:

    In this study we find that the function of BRCA1 inhibits the microtubule nucleation function of centrosomes. In particular, cells in early S phase have quiescent centrosomes due to BRCA1 activity, which inhibits the association of gamma-tubulin with centrosomes. We find that modification of either of two specific lysine residues (Lys-48 and Lys-344) of gamma-tubulin, a known substrate for BRCA1-dependent ubiquitination activity, led to centrosome hyperactivity. Interestingly, mutation of gamma-tubulin lysine 344 had a minimal effect on centrosome number but a profound effect on microtubule nucleation function, indicating that the processes regulating centrosome duplication and microtubule nucleation are distinct. Using an in vitro aster formation assay, we found that BRCA1-dependent ubiquitination activity directly inhibits microtubule nucleation by centrosomes. Mutant BRCA1 Protein that was inactive as a ubiquitin ligase did not inhibit aster formation by the centrosome. Further, a BRCA1 carboxy-terminal truncation mutant that was an active ubiquitin ligase lacked domains critical for the inhibition of centrosome function. These experiments reveal an important new functional assay regulated by the BRCA1-dependent ubiquitin ligase, and the results suggest that the loss of this BRCA1 activity could cause the centrosome hypertrophy and subsequent aneuploidy typically found in breast cancers.

  • BRCA1 Protein is linked to the rna polymerase ii holoenzyme complex via rna helicase a
    Nature Genetics, 1998
    Co-Authors: Stephen F Anderson, Toshihiro Nakajima, Brian P Schlegel, Eric S Wolpin, Jeffrey D Parvin
    Abstract:

    The breast cancer specific tumour suppressor Protein, BRCA1 (refs 1,2), activates transcription when linked with a DNA-binding domain3,4 and is a component of the RNA polymerase II (Pol II) holoenzyme5,6. We show here that RNA helicase A (RHA) Protein7,8 links BRCA1 to the holoenzyme complex. The region of BRCA1 which interacts with RHA and, thus, the holoenzyme complex, corresponds to subregions of the BRCT domain of BRCA1 ( ref. 9). This interaction was shown to occur in yeast nuclei, and expression in human cells of a truncated RHA molecule which retains binding to BRCA1 inhibited transcriptional activation mediated by the BRCA1 carboxy terminus. These data are the first to identify a specific Protein interaction with the BRCA1 C-terminal domain and are consistent with the model that BRCA1 functions as a transcriptional coactivator.

  • BRCA1 is a component of the rna polymerase ii holoenzyme
    Proceedings of the National Academy of Sciences of the United States of America, 1997
    Co-Authors: Ralph Scully, Richard A Young, David M. Chao, Stephen Anderson, Wanjiang Wei, David M Livingston, Jeffrey D Parvin
    Abstract:

    The familial breast–ovarian tumor suppressor gene product BRCA1 was found to be a component of the RNA polymerase II holoenzyme by several criteria. BRCA1 was found to copurify with the holoenzyme over multiple chromatographic steps. Other tested transcription activators that could potentially contact the holoenzyme were not stably associated with the holoenzyme as determined by copurification. Antibody specific for the holoenzyme component hSRB7 specifically purifies BRCA1. Immunopurification of BRCA1 complexes also specifically purifies transcriptionally active RNA polymerase II and transcription factors TFIIF, TFIIE, and TFIIH. Moreover, a BRCA1 domain, which is deleted in about 90% of clinically relevant mutations, participates in binding to the holoenzyme complex in cells. These data are consistent with recent data identifying transcription activation domains in the BRCA1 Protein and link the BRCA1 tumor suppressor Protein with the transcription process as a holoenzyme-bound Protein.

Adisorn Ratanaphan - One of the best experts on this subject based on the ideXlab platform.

  • cellular responses of BRCA1 defective hcc1937 breast cancer cells induced by the antimetastasis ruthenium ii arene compound rapta t
    Apoptosis, 2019
    Co-Authors: Tidarat Nhukeaw, Khwanjira Hongthong, Paul J Dyson, Adisorn Ratanaphan
    Abstract:

    An organometallic ruthenium(II) arene compound, Ru(η6-toluene)(PTA)Cl2 (PTA = 1,3,5-triaza-7-phosphaadamantane), termed RAPTA-T, exerts promising antimetastatic properties. In this study, the effects of RAPTA-T on BRCA1-defective HCC1937 breast cancer cells have been investigated, and compared to its effects on BRCA1-competent MCF-7 breast cancer cells. RAPTA-T showed a very low cytotoxicity against both tested cells. Ruthenium is found mostly in the cytoplasmic compartment of both cells. Flow cytometric analysis reveals that the compound arrests the growth of both cells by triggering the G2/M phase that led to the induction of apoptosis. At equimolar concentrations, RAPTA-T causes much more cellular BRCA1 damage in HCC1937 than in MCF-7 cells, suppressing the expression of BRCA1 mRNA in both cell lines with the subsequent down-regulation of the BRCA1 Protein. Interestingly, RAPTA-T exhibits an approximately fivefold greater ability to suppress the expression of the BRCA1 Protein in HCC1937 than in MCF-7 cells. These data provide insights into the molecular mechanisms by which RAPTA-T exerts its effects on BRCA1-associated breast cancer cells.

  • differential cytotoxicity cellular uptake apoptosis and inhibition of BRCA1 expression of BRCA1 defective and sporadic breast cancer cells induced by an anticancer ruthenium ii arene compound rapta ea1
    Anti-cancer Agents in Medicinal Chemistry, 2017
    Co-Authors: Adisorn Ratanaphan, Tidarat Nhukeaw, Khwanjira Hongthong, Paul J Dyson
    Abstract:

    Background: The RAPTA-EA1 complex [ruthenium(II)-arene 1,3,5-triaza-7-phosphaadamantane (pta) complex with an arene-tethered ethacrynic acid ligand] has been reported to overcome drug resistance that developed due to the current use of platinum-based treatments. However, the exact mechanism of action of RAPTA-EA1 remains largely unexplored and unknown. Objective: Here we have further studied the effect of RAPTA-EA1 on BRCA1-defective HCC1937 breast cancer cells and compared its effects on BRCA1-competent MCF-7 breast cancer cells. Method: HCC1937 and MCF-7 breast cancer cells were treated with the RAPTA-EA1 complex. The cytotoxicity of ruthenium-induced cells was evaluated by a MTT assay. Cellular uptake of ruthenium was determined by ICP-MS. Cell cycle and apoptosis were assessed using a flow cytometer. Expression of BRCA1 mRNA and its encoded Protein was quantitated by a real-time RT-PCR and Western blotting. Results: Differences in cytotoxicity were correlated with the differential accumulations of ruthenium and the induction of apoptosis. The ruthenium complex caused dramatically more damage to the BRCA1 gene in the BRCA1-defective HCC1937 cells than to the BRCA1-competent MCF-7 cells. It decreased the expression of BRCA1 mRNA in the BRCA1-competent cells, while in contrast, its expression increased in the BRCA1-defective cells. However, the expression of the BRCA1 Protein was significantly reduced in both types of breast cancer cells. Conclusion: The results presented here have demonstrated a differential cellular response for the BRCA1-defective and BRCA1-competent breast cancer cells to RAPTA-EA1. These findings have provided more insight into the actions and development of the ruthenium-based compounds for use for the treatment of breast cancer.

Paul J Dyson - One of the best experts on this subject based on the ideXlab platform.

  • cellular responses of BRCA1 defective hcc1937 breast cancer cells induced by the antimetastasis ruthenium ii arene compound rapta t
    Apoptosis, 2019
    Co-Authors: Tidarat Nhukeaw, Khwanjira Hongthong, Paul J Dyson, Adisorn Ratanaphan
    Abstract:

    An organometallic ruthenium(II) arene compound, Ru(η6-toluene)(PTA)Cl2 (PTA = 1,3,5-triaza-7-phosphaadamantane), termed RAPTA-T, exerts promising antimetastatic properties. In this study, the effects of RAPTA-T on BRCA1-defective HCC1937 breast cancer cells have been investigated, and compared to its effects on BRCA1-competent MCF-7 breast cancer cells. RAPTA-T showed a very low cytotoxicity against both tested cells. Ruthenium is found mostly in the cytoplasmic compartment of both cells. Flow cytometric analysis reveals that the compound arrests the growth of both cells by triggering the G2/M phase that led to the induction of apoptosis. At equimolar concentrations, RAPTA-T causes much more cellular BRCA1 damage in HCC1937 than in MCF-7 cells, suppressing the expression of BRCA1 mRNA in both cell lines with the subsequent down-regulation of the BRCA1 Protein. Interestingly, RAPTA-T exhibits an approximately fivefold greater ability to suppress the expression of the BRCA1 Protein in HCC1937 than in MCF-7 cells. These data provide insights into the molecular mechanisms by which RAPTA-T exerts its effects on BRCA1-associated breast cancer cells.

  • differential cytotoxicity cellular uptake apoptosis and inhibition of BRCA1 expression of BRCA1 defective and sporadic breast cancer cells induced by an anticancer ruthenium ii arene compound rapta ea1
    Anti-cancer Agents in Medicinal Chemistry, 2017
    Co-Authors: Adisorn Ratanaphan, Tidarat Nhukeaw, Khwanjira Hongthong, Paul J Dyson
    Abstract:

    Background: The RAPTA-EA1 complex [ruthenium(II)-arene 1,3,5-triaza-7-phosphaadamantane (pta) complex with an arene-tethered ethacrynic acid ligand] has been reported to overcome drug resistance that developed due to the current use of platinum-based treatments. However, the exact mechanism of action of RAPTA-EA1 remains largely unexplored and unknown. Objective: Here we have further studied the effect of RAPTA-EA1 on BRCA1-defective HCC1937 breast cancer cells and compared its effects on BRCA1-competent MCF-7 breast cancer cells. Method: HCC1937 and MCF-7 breast cancer cells were treated with the RAPTA-EA1 complex. The cytotoxicity of ruthenium-induced cells was evaluated by a MTT assay. Cellular uptake of ruthenium was determined by ICP-MS. Cell cycle and apoptosis were assessed using a flow cytometer. Expression of BRCA1 mRNA and its encoded Protein was quantitated by a real-time RT-PCR and Western blotting. Results: Differences in cytotoxicity were correlated with the differential accumulations of ruthenium and the induction of apoptosis. The ruthenium complex caused dramatically more damage to the BRCA1 gene in the BRCA1-defective HCC1937 cells than to the BRCA1-competent MCF-7 cells. It decreased the expression of BRCA1 mRNA in the BRCA1-competent cells, while in contrast, its expression increased in the BRCA1-defective cells. However, the expression of the BRCA1 Protein was significantly reduced in both types of breast cancer cells. Conclusion: The results presented here have demonstrated a differential cellular response for the BRCA1-defective and BRCA1-competent breast cancer cells to RAPTA-EA1. These findings have provided more insight into the actions and development of the ruthenium-based compounds for use for the treatment of breast cancer.

Tidarat Nhukeaw - One of the best experts on this subject based on the ideXlab platform.

  • cellular responses of BRCA1 defective hcc1937 breast cancer cells induced by the antimetastasis ruthenium ii arene compound rapta t
    Apoptosis, 2019
    Co-Authors: Tidarat Nhukeaw, Khwanjira Hongthong, Paul J Dyson, Adisorn Ratanaphan
    Abstract:

    An organometallic ruthenium(II) arene compound, Ru(η6-toluene)(PTA)Cl2 (PTA = 1,3,5-triaza-7-phosphaadamantane), termed RAPTA-T, exerts promising antimetastatic properties. In this study, the effects of RAPTA-T on BRCA1-defective HCC1937 breast cancer cells have been investigated, and compared to its effects on BRCA1-competent MCF-7 breast cancer cells. RAPTA-T showed a very low cytotoxicity against both tested cells. Ruthenium is found mostly in the cytoplasmic compartment of both cells. Flow cytometric analysis reveals that the compound arrests the growth of both cells by triggering the G2/M phase that led to the induction of apoptosis. At equimolar concentrations, RAPTA-T causes much more cellular BRCA1 damage in HCC1937 than in MCF-7 cells, suppressing the expression of BRCA1 mRNA in both cell lines with the subsequent down-regulation of the BRCA1 Protein. Interestingly, RAPTA-T exhibits an approximately fivefold greater ability to suppress the expression of the BRCA1 Protein in HCC1937 than in MCF-7 cells. These data provide insights into the molecular mechanisms by which RAPTA-T exerts its effects on BRCA1-associated breast cancer cells.

  • differential cytotoxicity cellular uptake apoptosis and inhibition of BRCA1 expression of BRCA1 defective and sporadic breast cancer cells induced by an anticancer ruthenium ii arene compound rapta ea1
    Anti-cancer Agents in Medicinal Chemistry, 2017
    Co-Authors: Adisorn Ratanaphan, Tidarat Nhukeaw, Khwanjira Hongthong, Paul J Dyson
    Abstract:

    Background: The RAPTA-EA1 complex [ruthenium(II)-arene 1,3,5-triaza-7-phosphaadamantane (pta) complex with an arene-tethered ethacrynic acid ligand] has been reported to overcome drug resistance that developed due to the current use of platinum-based treatments. However, the exact mechanism of action of RAPTA-EA1 remains largely unexplored and unknown. Objective: Here we have further studied the effect of RAPTA-EA1 on BRCA1-defective HCC1937 breast cancer cells and compared its effects on BRCA1-competent MCF-7 breast cancer cells. Method: HCC1937 and MCF-7 breast cancer cells were treated with the RAPTA-EA1 complex. The cytotoxicity of ruthenium-induced cells was evaluated by a MTT assay. Cellular uptake of ruthenium was determined by ICP-MS. Cell cycle and apoptosis were assessed using a flow cytometer. Expression of BRCA1 mRNA and its encoded Protein was quantitated by a real-time RT-PCR and Western blotting. Results: Differences in cytotoxicity were correlated with the differential accumulations of ruthenium and the induction of apoptosis. The ruthenium complex caused dramatically more damage to the BRCA1 gene in the BRCA1-defective HCC1937 cells than to the BRCA1-competent MCF-7 cells. It decreased the expression of BRCA1 mRNA in the BRCA1-competent cells, while in contrast, its expression increased in the BRCA1-defective cells. However, the expression of the BRCA1 Protein was significantly reduced in both types of breast cancer cells. Conclusion: The results presented here have demonstrated a differential cellular response for the BRCA1-defective and BRCA1-competent breast cancer cells to RAPTA-EA1. These findings have provided more insight into the actions and development of the ruthenium-based compounds for use for the treatment of breast cancer.

Konrad H Stopsack - One of the best experts on this subject based on the ideXlab platform.

  • tumor Protein expression of the dna repair gene BRCA1 and lethal prostate cancer
    Carcinogenesis, 2020
    Co-Authors: Konrad H Stopsack, Travis Gerke, Piotr Zareba, Andreas Pettersson, Dipanjan Chowdhury, Ericka M Ebot, Richard Flavin
    Abstract:

    DNA repair genes are commonly altered in metastatic prostate cancer, but BRCA1 mutations are rare. Preliminary studies suggest that higher tumor expression of the BRCA1 Protein may be associated with worse prognosis. We undertook a prospective study among men with prostate cancer in the Health Professionals Follow-up Study and evaluated BRCA1 via immunohistochemical staining on tissue microarrays. BRCA1 was expressed in 60 of 589 tumors. Prevalence of BRCA1 positivity was 43% in the 14 men with metastases at diagnosis compared with 9% in non-metastatic tumors [difference, 33 percentage points; 95% confidence interval (CI), 7-59]. BRCA1-positive tumors had 2.16-fold higher Ki-67 proliferative indices (95% CI, 1.18-3.95), higher tumor aneuploidy as predicted from whole-transcriptome profiling, and higher Gleason scores. Among the 575 patients with non-metastatic disease at diagnosis, we evaluated the association between BRCA1 expression and development of lethal disease (metastasis or cancer-specific death, 69 events) during long-term follow-up (median, 18.3 years). A potential weak association of BRCA1 positivity with lethal disease (hazard ratio, 1.61; 95% CI, 0.82-3.15) was attenuated when adjusting for age, Gleason score and clinical stage (hazard ratio, 1.11; 95% CI, 0.54-2.29). In summary, BRCA1 Protein expression is a feature of more proliferative and more aneuploid prostate tumors and is more common in metastatic disease. While not well suited as a prognostic biomarker in primary prostate cancer, BRCA1 Protein expression may be most relevant in advanced disease.

  • tumor Protein expression of the dna repair gene BRCA1 and lethal prostate cancer
    Carcinogenesis, 2020
    Co-Authors: Konrad H Stopsack, Travis Gerke, Piotr Zareba, Andreas Pettersson, Dipanjan Chowdhury, Ericka M Ebot, Richard Flavin
    Abstract:

    DNA repair genes are commonly altered in metastatic prostate cancer, but BRCA1 mutations are rare. Preliminary studies suggest that higher tumor expression of the BRCA1 Protein may be associated with worse prognosis. We undertook a prospective study among men with prostate cancer in the Health Professionals Follow-up Study and evaluated BRCA1 via immunohistochemical staining on tissue microarrays. BRCA1 was expressed in 60 of 589 tumors. Prevalence of BRCA1 positivity was 43% in the 14 men with metastases at diagnosis compared to 9% in non-metastatic tumors (difference, 33 percentage points; 95% CI, 7 to 59). BRCA1-positive tumors had 2.16-fold higher Ki-67 proliferative indices (95% CI, 1.18 to 3.95), higher tumor aneuploidy as predicted from whole-transcriptome profiling, and higher Gleason scores. Among the 575 patients with non-metastatic disease at diagnosis, we evaluated the association between BRCA1 expression and development of lethal disease (metastasis or cancer-specific death, 69 events) during long-term follow-up (median, 18.3 years). A potential weak association of BRCA1 positivity with lethal disease (hazard ratio, 1.61; 95% CI, 0.82 to 3.15) was attenuated when adjusting for age, Gleason score, and clinical stage (hazard ratio, 1.11; 95% CI, 0.54 to 2.29). In summary, BRCA1 Protein expression is a feature of more proliferative and more aneuploid prostate tumors and is more common in metastatic disease. While not well suited as a prognostic biomarker in primary prostate cancer, BRCA1 Protein expression may be most relevant in advanced disease.