Mutational activation of the BRAF proto-oncogene in melanocytes reliably produces benign nevi (pi... more Mutational activation of the BRAF proto-oncogene in melanocytes reliably produces benign nevi (pigmented 'moles'), yet the same change is the most common driver mutation in melanoma. The reason nevi stop growing, and do not progress to melanoma, is widely attributed to a cell-autonomous process of 'oncogene-induced senescence'. Using a mouse model of Brafdriven nevus formation, analyzing both proliferative dynamics and single-cell gene expression, we found no evidence that nevus cells are senescent, either compared with other skin cells, or other melanocytes. We also found that nevus size distributions could not be fit by any simple cellautonomous model of growth arrest, yet were easily fit by models based on collective cell behavior, for example in which arresting cells release an arrest-promoting factor. We suggest that nevus growth arrest is more likely related to the cell interactions that mediate size control in normal tissues, than to any cell-autonomous, 'oncogene-induced' program of senescence.
Mutational activation of the BRAF proto-oncogene in melanocytes reliably produces benign nevi (pi... more Mutational activation of the BRAF proto-oncogene in melanocytes reliably produces benign nevi (pigmented ‘moles’), yet the same change is the most common driver mutation in melanoma. The reason nevi stop growing, and do not progress to melanoma, is widely attributed to a cell-autonomous process of ‘oncogene-induced senescence’. Using a mouse model of Braf-driven nevus formation, analyzing both proliferative dynamics and single-cell gene expression, we found no evidence that nevus cells are senescent, either compared with other skin cells, or other melanocytes. We also found that nevus size distributions could not be fit by any simple cell-autonomous model of growth arrest, yet were easily fit by models based on collective cell behavior, for example in which arresting cells release an arrest-promoting factor. We suggest that nevus growth arrest is more likely related to the cell interactions that mediate size control in normal tissues, than to any cell-autonomous, ‘oncogene-induced’ ...
Genes and pathways that allow cells to cope with oncogene-induced stress represent selective canc... more Genes and pathways that allow cells to cope with oncogene-induced stress represent selective cancer therapeutic targets that remain largely undiscovered. In this study, we identify a RhoJ signaling pathway that is a selective therapeutic target for BRAF mutant cells. RhoJ deletion in BRAF mutant melanocytes modulates the expression of the pro-apoptotic protein BAD as well as genes involved in cellular metabolism, impairing nevus formation, cellular transformation, and metastasis. Short-term treatment of nascent melanoma tumors with PAK inhibitors that block RhoJ signaling halts the growth of BRAF mutant melanoma tumors in vivo and induces apoptosis in melanoma cells in vitro via a BAD-dependent mechanism. As up to 50% of BRAF mutant human melanomas express high levels of RhoJ, these studies nominate the RhoJ-BAD signaling network as a therapeutic vulnerability for fledgling BRAF mutant human tumors.
Melanomas accumulate a high burden of mutations that could potentially generate neoantigens, yet ... more Melanomas accumulate a high burden of mutations that could potentially generate neoantigens, yet somehow suppress the immune response to facilitate continued growth. In this study, we identify a subset of human melanomas that have loss-of-function mutations in ATR, a kinase that recognizes and repairs UV-induced DNA damage and is required for cellular proliferation. ATR mutant tumors exhibit both the accumulation of multiple mutations and the altered expression of inflammatory genes, resulting in decreased T cell recruitment and increased recruitment of macrophages known to spur tumor invasion. Taken together, these studies identify a mechanism by which melanoma cells modulate the immune microenvironment to promote continued growth.
Renal cell carcinoma (RCC) is a heterogeneous disease with resistance to systemic chemotherapy. E... more Renal cell carcinoma (RCC) is a heterogeneous disease with resistance to systemic chemotherapy. Elevated expression of multiple drug resistance (MDR) has been suggested to be one of the mechanisms for this resistance. Here, we provide an alternative mechanism to explain RCC's resistance to chemotherapy-induced apoptosis. Never-in mitosis A-related protein kinase 1 (Nek1) plays an important role in DNA damage response and proper checkpoint activation. The association of Nek1 with the voltage-dependent anion channel (VDAC1) is a critical determinant of cell survival following DNA-damaging treatment. We report here that Nek1 is highly expressed in RCC tumor and cultured RCC cells compared to that of normal renal tubular epithelial cells (RTE). The association between Nek1 and VDAC1 is genotoxic dependent: prolonged Nek1/VDAC1 dissociation will lead to VDAC1 dephosphorylation and initiate apoptosis. Down-regulation of Nek1 expression in RCC cells enhanced their sensitivity to DNA-da...
BRCA1 encodes a tumor suppressor that is mutated in familial breast and ovarian cancers. Here, it... more BRCA1 encodes a tumor suppressor that is mutated in familial breast and ovarian cancers. Here, it is shown that BRCA1 interacts in vitro and in vivo with hRad50, which forms a complex with hMre11 and p95/nibrin. Upon irradiation, BRCA1 was detected in discrete foci in the nucleus, which colocalize with hRad50. Formation of irradiation-induced foci positive for BRCA1, hRad50, hMre11, or p95 was dramatically reduced in HCC/1937 breast cancer cells carrying a homozygous mutation in BRCA1 but was restored by transfection of wild-type BRCA1 . Ectopic expression of wild-type, but not mutated, BRCA1 in these cells rendered them less sensitive to the DNA damage agent, methyl methanesulfonate. These data suggest that BRCA1 is important for the cellular responses to DNA damage that are mediated by the hRad50-hMre11-p95 complex.
Proceedings of the National Academy of Sciences, 1999
Trip230 is a novel coactivator of the thyroid hormone receptor that is negatively regulated by th... more Trip230 is a novel coactivator of the thyroid hormone receptor that is negatively regulated by the retinoblastoma tumor-suppressor protein. In an examination of its subcellular distribution, Trip230 localized predominantly to the vicinity of the Golgi instead of the nucleus, as other nuclear hormone receptor coactivators. Using a series of deletion mutants, a critical region identified for Golgi area targeting coincided with a previously defined thyroid hormone receptor-binding domain of Trip230. During cell cycle progression, the expression level of Trip230 is constant and a significant portion is imported into the nucleus at S phase. Within an hour of treating cells with T3, Trip230 immunofluorescence transiently colocalized with TR in prominent subnuclear structures. T3-dependent nuclear import of Trip230 does not require new protein synthesis. Coincident with T3 treatment and nuclear import, newly phosphorylated residue(s) appeared in Trip230, suggesting that phosphorylation may...
tsglOl was identified as a tumor susceptibifity gene by homozygous functional inactivation ofalle... more tsglOl was identified as a tumor susceptibifity gene by homozygous functional inactivation ofallelic loci mouse 3T3 fibroblasts. The human homologue was mapped at chromosome llplS.lâ€"2 and found to have intragenic deletion In 7 of 15 breast cancer specimens. To further confirm the relevance of defects in this gene to breast cancer, antibodies specific for the putative gene product were prepared and used to identify cellular TSG1O1protein. The antibodies recognized a 46-kDa protein in human retinoblastoma WERI-27 cells labeled with (35S)methionine.This protein was not detected with preimmune sera, In cell fractionation studies, the 46-kDa protein cofractionating with glutathione S-transferase was found mainly in the cytoplasm. Similarly, when cells were Immunostained with anti-TSG1O1 antibodies, fluorescence was localized in the cytoplasm of most of the cells. A full-size 46-kDa TSG1O1protein was detected in a panel of 10 breast cancer cell lines and 2 normal breast epithelial ...
Background: NEK1, the first mammalian ortholog of the fungal protein kinase never-in-mitosis A (N... more Background: NEK1, the first mammalian ortholog of the fungal protein kinase never-in-mitosis A (NIMA), is involved early in the DNA damage sensing/repair pathway. A defect in DNA repair in NEK1-deficient cells is suggested by persistence of DNA double strand breaks after low dose ionizing radiation (IR). NEK1-deficient cells also fail to activate the checkpoint kinases CHK1 and CHK2, and fail to arrest properly at G1/S or G2/M-phase checkpoints after DNA damage. Results: We show here that NEK1-deficient cells suffer major errors in mitotic chromosome segregation and cytokinesis, and become aneuploid. These NEK1-deficient cells transform, acquire the ability to grow in anchorageindependent conditions, and form tumors when injected into syngeneic mice. Genomic instability is also manifest in NEK1 +/-mice, which late in life develop lymphomas with a much higher incidence than wild type littermates. Conclusion: NEK1 is required for the maintenance of genome stability by acting at multiple junctures, including control of chromosome stability.
BRCA2 is a breast tumor suppressor with a potential function in the cellular response to DNA dama... more BRCA2 is a breast tumor suppressor with a potential function in the cellular response to DNA damage. BRCA2 binds to Rad51 through its BRC repeats. In support of the biological significance of this interaction, we found that the complex of BRCA2 and Rad51 in breast cancer MCF-7 cells was diminished upon conditional expression of a wild-type, but not a mutated, BRC4 repeat using the tetracycline-inducible system. Cells expressing a wild-type BRC4 repeat showed hypersensitivity to ␥-irradiation, an inability to form Rad51 radiationinduced foci, and a failure of radiation-induced G 2 /M, but not G 1 /S, checkpoint control. These results strongly suggest that the interaction between BRCA2 and Rad51 mediated by BRC repeats is critical for the cellular response to DNA damage. BRCA2 was cloned based on an analysis of mutations in families predisposed to breast cancer showing that a large percentage of the kindred had alterations within this locus (1, 2). The expression pattern of BRCA2 is remarkably similar to that of BRCA1 (3-5), with highest levels in the testis, thymus, and ovaries (5). At the cellular level, expression is regulated in a cell-cycle dependent manner and peak expression of BRCA2 mRNA is found in S phase (6). These results suggest BRCA2 may participate in regulating cell proliferation. Recent studies indicate that BRCA2 is important for the cellular response to DNA damage. Brca2-null mouse embryos are nonviable at a very early stage of development and blastocysts derived from these embryos are very sensitive to ␥-irradiation (7). Mouse embryonic fibroblasts predicted to express BRCA2 that is C-terminally truncated at amino acid 1492 also demonstrated sensitivity to DNA damaging agents, particularly methyl methanesulfonate and UV light (8). Furthermore, Capan-1, a human pancreatic cancer line, that expresses a 220-kDa C-terminally truncated BRCA2 protein, is hypersensitive to a panel of DNA damaging agents (9). Importantly, ectopic expression of wild-type, but not mutated, BRCA2 in Capan-1 cells restores resistance to treatment with MMS (10). These results provided convincing evidence that BRCA2 plays a critical role in the DNA repair process. Interestingly, BRCA2 was shown to interact with Rad51 (7, 10-12), a key protein in DNA recombinational repair. Human Rad51 encodes a 40-kDa protein with a structure related to the * This work was supported by Grants CA58183 and CA30195 from the NCI (to W. H. L.
Background: Helicase SUV3, polynucleotide phosphorylase (PNPase), or mitochondrial poly(A) polyme... more Background: Helicase SUV3, polynucleotide phosphorylase (PNPase), or mitochondrial poly(A) polymerases (mtPAP) have individual activity in regulating mitochondrial mRNA (mt-mRNA)-polyadenylated (poly(A)) tails. Results: SUV3 bridges PNPase and mtPAP to form a transient complex in modulating mt-mRNA poly(A) tail length depending on the mitochondrial matrix P i level. Conclusion: mt-mRNA poly(A) tail length is modulated by a SUV3⅐PNPase⅐mtPAP complex. Significance: Mitochondrial energy status affects mt-mRNA poly(A) tail length. Mammalian mitochondrial mRNA (mt-mRNA) transcripts are polyadenylated at the 3 end with different lengths. The SUV3⅐PNPase complex and mtPAP have been shown to degrade and polyadenylate mt mRNA, respectively. How these two opposite actions are coordinated to modulate mt-mRNA poly(A) lengths is of interest to pursue. Here, we demonstrated that a fraction of the SUV3⅐PNPase complex interacts with mitochondrial polyadenylation polymerase (mtPAP) under low mitochondrial matrix inorganic phosphate (P i) conditions. In vitro binding experiments using purified proteins suggested that SUV3 binds to mtPAP through the N-terminal region around amino acids 100-104, distinctive from the C-terminal region around amino acids 510-514 of SUV3 for PNPase binding. mtPAP does not interact with PNPase directly, and SUV3 served as a bridge capable of simultaneously binding with mtPAP and PNPase. The complex consists of a SUV3 dimer, a mtPAP dimer, and a PNPase trimer, based on the molecular sizing experiments. Mechanistically, SUV3 provides a robust single strand RNA binding domain to enhance the polyadenylation activity of mtPAP. Furthermore, purified SUV3⅐ PNPase⅐mtPAP complex is capable of lengthening or shortening the RNA poly(A) tail lengths in low or high P i /ATP ratios, respectively. Consistently, the poly(A) tail lengths of mt-mRNA transcripts can be lengthened or shortened by altering the mitochondrial matrix P i levels via selective inhibition of the electron transport chain or ATP synthase, respectively. Taken together, these results suggested that SUV3⅐PNPase⅐mtPAP form a transient complex to modulate mt-mRNA poly(A) tail lengths in response to cellular energy changes.
BRCA1 is critical for the maintenance of genomic stability, in part through its interaction with ... more BRCA1 is critical for the maintenance of genomic stability, in part through its interaction with the Rad50⅐Mre11⅐Nbs1 complex, which occupies a central role in DNA double strand break repair mediated by nonhomologous end joining (NHEJ) and homologous recombination. BRCA1 has been shown to be required for homology-directed recombination repair. However, the role of BRCA1 in NHEJ, a critical pathway for the repair of double strand breaks and genome stability in mammalian cells, remains elusive. Here, we established a pair of mouse embryonic fibroblasts (MEFs) derived from 9.5-day-old embryos with genotypes Brca1 ؉/؉ : p53 ؊/؊ or Brca1 ؊/؊ :p53 ؊/؊. The Brca1 ؊/؊ :p53 ؊/؊ MEFs appear to be extremely sensitive to ionizing radiation. The contribution of BRCA1 in NHEJ was evaluated in these cells using three different assay systems. First, transfection of a linearized plasmid in which expression of the reporter gene required precise end joining indicated that Brca1 ؊/؊ MEFs display a moderate deficiency when compared with Brca1 ؉/؉ cells. Second, using a retrovirus infection assay dependent on NHEJ, a 5-10-fold reduction in retroviral integration efficiency was observed in Brca1 ؊/؊ MEFs when compared with the Brca1 ؉/؉ MEFs. Third, Brca1 ؊/؊ MEFs exhibited a 50-100-fold deficiency in microhomology-mediated endjoining activity of a defined chromosomal DNA double strand break introduced by a rare cutting endonuclease I-SceI. These results provide evidence that Brca1 has an essential role in microhomology-mediated end joining and suggest a novel molecular basis for its caretaker role in the maintenance of genome integrity.
Background: SUV3 helicase is an essential component of mitochondrial degradosome. Results: Using ... more Background: SUV3 helicase is an essential component of mitochondrial degradosome. Results: Using specific genetic mutants, SUV3 was demonstrated to participate in mtDNA replication, which requires ATPase activity and the C-terminal conserved region. Conclusion: SUV3 plays two distinctive roles in maintaining mtDNA stability and RNA turnover. Significance: This study suggests a direct role of SUV3 in maintaining mtDNA stability, paving a foundation for biochemical investigation. Yeast SUV3 is a nuclear encoded mitochondrial RNA helicase that complexes with an exoribonuclease, DSS1, to function as an RNA degradosome. Inactivation of SUV3 leads to mitochondrial dysfunctions, such as respiratory deficiency; accumulation of aberrant RNA species, including excised group I introns; and loss of mitochondrial DNA (mtDNA). Although intron toxicity has long been speculated to be the major reason for the observed phenotypes, direct evidence to support or refute this theory is lacking. Moreover, it remains unknown whether SUV3 plays a direct role in mtDNA maintenance independently of its degradosome activity. In this paper, we address these questions by employing an inducible knockdown system in Saccharomyces cerevisiae with either normal or intronless mtDNA background. Expressing mutants defective in ATPase (K245A) or RNA binding activities (V272L or ⌬CC, which carries an 8-amino acid deletion at the C-terminal conserved region) resulted in not only respiratory deficiencies but also loss of mtDNA under normal mtDNA background. Surprisingly, V272L, but not other mutants, can rescue the said deficiencies under intronless background. These results provide genetic evidence supporting the notion that the functional requirements of SUV3 for degradosome activity and maintenance of mtDNA stability are separable. Furthermore, V272L mutants and wild-type SUV3 associated with an active mtDNA replication origin and facilitated mtDNA replication, whereas K245A and ⌬CC failed to support mtDNA replication. These results indicate a direct role of SUV3 in maintaining mitochondrial genome stability that is independent of intron turnover but requires the intact ATPase activity and the CC conserved region.
RAD51 recombinase activity plays a critical role for cancer cell proliferation and survival, and ... more RAD51 recombinase activity plays a critical role for cancer cell proliferation and survival, and often contributes to drug-resistance. Abnormally elevated RAD51 function and hyperactive homologous recombination (HR) rates have been found in a panel of cancers, including breast cancer and chronic myeloid leukaemia (CML). Directly targeting RAD51 and attenuating the deregulated RAD51 activity has therefore been proposed as an alternative and supplementary strategy for cancer treatment. Here we show that a newly identified small molecule, IBR2, disrupts RAD51 multimerization, accelerates proteasome-mediated RAD51 protein degradation, reduces ionizing radiation-induced RAD51 foci formation, impairs HR, inhibits cancer cell growth and induces apoptosis. In a murine imatinib-resistant CML model bearing the T315I Bcr-abl mutation, IBR2, but not imatinib, significantly prolonged animal survival. Moreover, IBR2 effectively inhibits the proliferation of CD34 þ progenitor cells from CML patients resistant to known BCR-ABL inhibitors. Therefore, small molecule inhibitors of RAD51 may suggest a novel class of broad-spectrum therapeutics for difficult-to-treat cancers.
Suv3 of Saccharomyces cerevisiae has been classified as a mitochondrial RNA helicase. However, th... more Suv3 of Saccharomyces cerevisiae has been classified as a mitochondrial RNA helicase. However, the helicase domain in both yeast and human SUV3 varies considerably from the typical RNA helicase motifs. To investigate its enzymatic activities, a homogeneously purified preparation of SUV3 is required. Expression of a processed form of human SUV3 carrying an N-terminal deletion of 46 amino acids (SUV3DeltaN46) in a yeast suv3 null mutant, which otherwise fails to grow in a nonfermentable carbon source and forms petite colonies in glucose medium, rescues the null phenotype. Through a five-step chromatographic procedure, an 83 kDa SUV3DeltaN46 protein (SUV3-83) and a partially degraded 70 kDa product (SUV3-70) containing amino acids 68-685 were purified to homogeneity. Single- or double-stranded DNA and RNA stimulated ATPase activity of both proteins. SUV3-70, which retains core catalytic domains, can bind and unwind multiple duplex substrates of RNA and DNA with a 5'-3' directionality over a wide range of pH, while SUV3-83 has helicase activity at only acidic pH. ATP, but not nonhydrolyzable ATP, is essential for the unwinding activity, suggesting the requirement of the energy derived from ATP hydrolysis. Consistent with this notion, suv3 mutants containing alanine (A) or arginine (R) substitutions at the conserved lysine residue in the ATP binding site (K213) lost ATPase activity and also failed to unwind the substrates. Importantly, circular dichroism (CD) spectral analysis showed that SUV3-83, at pH 5.0, adopts a conformation similar to that of SUV3-70, suggesting a conformational change in SUV3-83 is required for its helicase activity. The physiological relevance of the multiple-substrate helicase activity of human SUV3 is discussed.
The 26S proteasome is a multisubunit complex responsible for degradation of ubiquitinated substra... more The 26S proteasome is a multisubunit complex responsible for degradation of ubiquitinated substrates, which plays a critical role in regulating various biological processes. To fully understand the function and regulation of the proteasome complex, an important step is to elucidate its subunit composition and posttranslational modifications. Toward this goal, a new affinity purification strategy has been developed using a derivative of the HB tag for rapid isolation of the human 26S proteasome complex for subsequent proteomic analysis. The purification of the complex is achieved from stable 293 cell lines expressing a HB-tagged proteasome subunit and by high-affinity streptavidin binding with TEV cleavage elution. The complete composition of the 26S proteasome complex, including recently assigned new subunits, is identified by LC-MS/MS. In addition, all known proteasome activator proteins and components involved in the ubiquitin-proteasome degradation pathway are identified. Aside from the subunit composition, the N-terminal modification and phosphorylation of the proteasome subunits have been characterized. Twelve novel phosphorylation sites from eight subunits have been identified, and N-terminal modifications are determined for 25 subunits, 12 of which have not been previously reported in mammals. We also observe different N-terminal processing of subunit Rpn2, which results in identification of two different N-termini of the protein. This work presents the first comprehensive characterization of the human 26S proteasome complex by affinity purification and tandem mass spectrometry. The detailed proteomic profiling obtained here is significant to future studies aiming at a complete understanding of the structure-function relationship of the human 26S proteasome complex.
Mutational activation of the BRAF proto-oncogene in melanocytes reliably produces benign nevi (pi... more Mutational activation of the BRAF proto-oncogene in melanocytes reliably produces benign nevi (pigmented 'moles'), yet the same change is the most common driver mutation in melanoma. The reason nevi stop growing, and do not progress to melanoma, is widely attributed to a cell-autonomous process of 'oncogene-induced senescence'. Using a mouse model of Brafdriven nevus formation, analyzing both proliferative dynamics and single-cell gene expression, we found no evidence that nevus cells are senescent, either compared with other skin cells, or other melanocytes. We also found that nevus size distributions could not be fit by any simple cellautonomous model of growth arrest, yet were easily fit by models based on collective cell behavior, for example in which arresting cells release an arrest-promoting factor. We suggest that nevus growth arrest is more likely related to the cell interactions that mediate size control in normal tissues, than to any cell-autonomous, 'oncogene-induced' program of senescence.
Mutational activation of the BRAF proto-oncogene in melanocytes reliably produces benign nevi (pi... more Mutational activation of the BRAF proto-oncogene in melanocytes reliably produces benign nevi (pigmented ‘moles’), yet the same change is the most common driver mutation in melanoma. The reason nevi stop growing, and do not progress to melanoma, is widely attributed to a cell-autonomous process of ‘oncogene-induced senescence’. Using a mouse model of Braf-driven nevus formation, analyzing both proliferative dynamics and single-cell gene expression, we found no evidence that nevus cells are senescent, either compared with other skin cells, or other melanocytes. We also found that nevus size distributions could not be fit by any simple cell-autonomous model of growth arrest, yet were easily fit by models based on collective cell behavior, for example in which arresting cells release an arrest-promoting factor. We suggest that nevus growth arrest is more likely related to the cell interactions that mediate size control in normal tissues, than to any cell-autonomous, ‘oncogene-induced’ ...
Genes and pathways that allow cells to cope with oncogene-induced stress represent selective canc... more Genes and pathways that allow cells to cope with oncogene-induced stress represent selective cancer therapeutic targets that remain largely undiscovered. In this study, we identify a RhoJ signaling pathway that is a selective therapeutic target for BRAF mutant cells. RhoJ deletion in BRAF mutant melanocytes modulates the expression of the pro-apoptotic protein BAD as well as genes involved in cellular metabolism, impairing nevus formation, cellular transformation, and metastasis. Short-term treatment of nascent melanoma tumors with PAK inhibitors that block RhoJ signaling halts the growth of BRAF mutant melanoma tumors in vivo and induces apoptosis in melanoma cells in vitro via a BAD-dependent mechanism. As up to 50% of BRAF mutant human melanomas express high levels of RhoJ, these studies nominate the RhoJ-BAD signaling network as a therapeutic vulnerability for fledgling BRAF mutant human tumors.
Melanomas accumulate a high burden of mutations that could potentially generate neoantigens, yet ... more Melanomas accumulate a high burden of mutations that could potentially generate neoantigens, yet somehow suppress the immune response to facilitate continued growth. In this study, we identify a subset of human melanomas that have loss-of-function mutations in ATR, a kinase that recognizes and repairs UV-induced DNA damage and is required for cellular proliferation. ATR mutant tumors exhibit both the accumulation of multiple mutations and the altered expression of inflammatory genes, resulting in decreased T cell recruitment and increased recruitment of macrophages known to spur tumor invasion. Taken together, these studies identify a mechanism by which melanoma cells modulate the immune microenvironment to promote continued growth.
Renal cell carcinoma (RCC) is a heterogeneous disease with resistance to systemic chemotherapy. E... more Renal cell carcinoma (RCC) is a heterogeneous disease with resistance to systemic chemotherapy. Elevated expression of multiple drug resistance (MDR) has been suggested to be one of the mechanisms for this resistance. Here, we provide an alternative mechanism to explain RCC's resistance to chemotherapy-induced apoptosis. Never-in mitosis A-related protein kinase 1 (Nek1) plays an important role in DNA damage response and proper checkpoint activation. The association of Nek1 with the voltage-dependent anion channel (VDAC1) is a critical determinant of cell survival following DNA-damaging treatment. We report here that Nek1 is highly expressed in RCC tumor and cultured RCC cells compared to that of normal renal tubular epithelial cells (RTE). The association between Nek1 and VDAC1 is genotoxic dependent: prolonged Nek1/VDAC1 dissociation will lead to VDAC1 dephosphorylation and initiate apoptosis. Down-regulation of Nek1 expression in RCC cells enhanced their sensitivity to DNA-da...
BRCA1 encodes a tumor suppressor that is mutated in familial breast and ovarian cancers. Here, it... more BRCA1 encodes a tumor suppressor that is mutated in familial breast and ovarian cancers. Here, it is shown that BRCA1 interacts in vitro and in vivo with hRad50, which forms a complex with hMre11 and p95/nibrin. Upon irradiation, BRCA1 was detected in discrete foci in the nucleus, which colocalize with hRad50. Formation of irradiation-induced foci positive for BRCA1, hRad50, hMre11, or p95 was dramatically reduced in HCC/1937 breast cancer cells carrying a homozygous mutation in BRCA1 but was restored by transfection of wild-type BRCA1 . Ectopic expression of wild-type, but not mutated, BRCA1 in these cells rendered them less sensitive to the DNA damage agent, methyl methanesulfonate. These data suggest that BRCA1 is important for the cellular responses to DNA damage that are mediated by the hRad50-hMre11-p95 complex.
Proceedings of the National Academy of Sciences, 1999
Trip230 is a novel coactivator of the thyroid hormone receptor that is negatively regulated by th... more Trip230 is a novel coactivator of the thyroid hormone receptor that is negatively regulated by the retinoblastoma tumor-suppressor protein. In an examination of its subcellular distribution, Trip230 localized predominantly to the vicinity of the Golgi instead of the nucleus, as other nuclear hormone receptor coactivators. Using a series of deletion mutants, a critical region identified for Golgi area targeting coincided with a previously defined thyroid hormone receptor-binding domain of Trip230. During cell cycle progression, the expression level of Trip230 is constant and a significant portion is imported into the nucleus at S phase. Within an hour of treating cells with T3, Trip230 immunofluorescence transiently colocalized with TR in prominent subnuclear structures. T3-dependent nuclear import of Trip230 does not require new protein synthesis. Coincident with T3 treatment and nuclear import, newly phosphorylated residue(s) appeared in Trip230, suggesting that phosphorylation may...
tsglOl was identified as a tumor susceptibifity gene by homozygous functional inactivation ofalle... more tsglOl was identified as a tumor susceptibifity gene by homozygous functional inactivation ofallelic loci mouse 3T3 fibroblasts. The human homologue was mapped at chromosome llplS.lâ€"2 and found to have intragenic deletion In 7 of 15 breast cancer specimens. To further confirm the relevance of defects in this gene to breast cancer, antibodies specific for the putative gene product were prepared and used to identify cellular TSG1O1protein. The antibodies recognized a 46-kDa protein in human retinoblastoma WERI-27 cells labeled with (35S)methionine.This protein was not detected with preimmune sera, In cell fractionation studies, the 46-kDa protein cofractionating with glutathione S-transferase was found mainly in the cytoplasm. Similarly, when cells were Immunostained with anti-TSG1O1 antibodies, fluorescence was localized in the cytoplasm of most of the cells. A full-size 46-kDa TSG1O1protein was detected in a panel of 10 breast cancer cell lines and 2 normal breast epithelial ...
Background: NEK1, the first mammalian ortholog of the fungal protein kinase never-in-mitosis A (N... more Background: NEK1, the first mammalian ortholog of the fungal protein kinase never-in-mitosis A (NIMA), is involved early in the DNA damage sensing/repair pathway. A defect in DNA repair in NEK1-deficient cells is suggested by persistence of DNA double strand breaks after low dose ionizing radiation (IR). NEK1-deficient cells also fail to activate the checkpoint kinases CHK1 and CHK2, and fail to arrest properly at G1/S or G2/M-phase checkpoints after DNA damage. Results: We show here that NEK1-deficient cells suffer major errors in mitotic chromosome segregation and cytokinesis, and become aneuploid. These NEK1-deficient cells transform, acquire the ability to grow in anchorageindependent conditions, and form tumors when injected into syngeneic mice. Genomic instability is also manifest in NEK1 +/-mice, which late in life develop lymphomas with a much higher incidence than wild type littermates. Conclusion: NEK1 is required for the maintenance of genome stability by acting at multiple junctures, including control of chromosome stability.
BRCA2 is a breast tumor suppressor with a potential function in the cellular response to DNA dama... more BRCA2 is a breast tumor suppressor with a potential function in the cellular response to DNA damage. BRCA2 binds to Rad51 through its BRC repeats. In support of the biological significance of this interaction, we found that the complex of BRCA2 and Rad51 in breast cancer MCF-7 cells was diminished upon conditional expression of a wild-type, but not a mutated, BRC4 repeat using the tetracycline-inducible system. Cells expressing a wild-type BRC4 repeat showed hypersensitivity to ␥-irradiation, an inability to form Rad51 radiationinduced foci, and a failure of radiation-induced G 2 /M, but not G 1 /S, checkpoint control. These results strongly suggest that the interaction between BRCA2 and Rad51 mediated by BRC repeats is critical for the cellular response to DNA damage. BRCA2 was cloned based on an analysis of mutations in families predisposed to breast cancer showing that a large percentage of the kindred had alterations within this locus (1, 2). The expression pattern of BRCA2 is remarkably similar to that of BRCA1 (3-5), with highest levels in the testis, thymus, and ovaries (5). At the cellular level, expression is regulated in a cell-cycle dependent manner and peak expression of BRCA2 mRNA is found in S phase (6). These results suggest BRCA2 may participate in regulating cell proliferation. Recent studies indicate that BRCA2 is important for the cellular response to DNA damage. Brca2-null mouse embryos are nonviable at a very early stage of development and blastocysts derived from these embryos are very sensitive to ␥-irradiation (7). Mouse embryonic fibroblasts predicted to express BRCA2 that is C-terminally truncated at amino acid 1492 also demonstrated sensitivity to DNA damaging agents, particularly methyl methanesulfonate and UV light (8). Furthermore, Capan-1, a human pancreatic cancer line, that expresses a 220-kDa C-terminally truncated BRCA2 protein, is hypersensitive to a panel of DNA damaging agents (9). Importantly, ectopic expression of wild-type, but not mutated, BRCA2 in Capan-1 cells restores resistance to treatment with MMS (10). These results provided convincing evidence that BRCA2 plays a critical role in the DNA repair process. Interestingly, BRCA2 was shown to interact with Rad51 (7, 10-12), a key protein in DNA recombinational repair. Human Rad51 encodes a 40-kDa protein with a structure related to the * This work was supported by Grants CA58183 and CA30195 from the NCI (to W. H. L.
Background: Helicase SUV3, polynucleotide phosphorylase (PNPase), or mitochondrial poly(A) polyme... more Background: Helicase SUV3, polynucleotide phosphorylase (PNPase), or mitochondrial poly(A) polymerases (mtPAP) have individual activity in regulating mitochondrial mRNA (mt-mRNA)-polyadenylated (poly(A)) tails. Results: SUV3 bridges PNPase and mtPAP to form a transient complex in modulating mt-mRNA poly(A) tail length depending on the mitochondrial matrix P i level. Conclusion: mt-mRNA poly(A) tail length is modulated by a SUV3⅐PNPase⅐mtPAP complex. Significance: Mitochondrial energy status affects mt-mRNA poly(A) tail length. Mammalian mitochondrial mRNA (mt-mRNA) transcripts are polyadenylated at the 3 end with different lengths. The SUV3⅐PNPase complex and mtPAP have been shown to degrade and polyadenylate mt mRNA, respectively. How these two opposite actions are coordinated to modulate mt-mRNA poly(A) lengths is of interest to pursue. Here, we demonstrated that a fraction of the SUV3⅐PNPase complex interacts with mitochondrial polyadenylation polymerase (mtPAP) under low mitochondrial matrix inorganic phosphate (P i) conditions. In vitro binding experiments using purified proteins suggested that SUV3 binds to mtPAP through the N-terminal region around amino acids 100-104, distinctive from the C-terminal region around amino acids 510-514 of SUV3 for PNPase binding. mtPAP does not interact with PNPase directly, and SUV3 served as a bridge capable of simultaneously binding with mtPAP and PNPase. The complex consists of a SUV3 dimer, a mtPAP dimer, and a PNPase trimer, based on the molecular sizing experiments. Mechanistically, SUV3 provides a robust single strand RNA binding domain to enhance the polyadenylation activity of mtPAP. Furthermore, purified SUV3⅐ PNPase⅐mtPAP complex is capable of lengthening or shortening the RNA poly(A) tail lengths in low or high P i /ATP ratios, respectively. Consistently, the poly(A) tail lengths of mt-mRNA transcripts can be lengthened or shortened by altering the mitochondrial matrix P i levels via selective inhibition of the electron transport chain or ATP synthase, respectively. Taken together, these results suggested that SUV3⅐PNPase⅐mtPAP form a transient complex to modulate mt-mRNA poly(A) tail lengths in response to cellular energy changes.
BRCA1 is critical for the maintenance of genomic stability, in part through its interaction with ... more BRCA1 is critical for the maintenance of genomic stability, in part through its interaction with the Rad50⅐Mre11⅐Nbs1 complex, which occupies a central role in DNA double strand break repair mediated by nonhomologous end joining (NHEJ) and homologous recombination. BRCA1 has been shown to be required for homology-directed recombination repair. However, the role of BRCA1 in NHEJ, a critical pathway for the repair of double strand breaks and genome stability in mammalian cells, remains elusive. Here, we established a pair of mouse embryonic fibroblasts (MEFs) derived from 9.5-day-old embryos with genotypes Brca1 ؉/؉ : p53 ؊/؊ or Brca1 ؊/؊ :p53 ؊/؊. The Brca1 ؊/؊ :p53 ؊/؊ MEFs appear to be extremely sensitive to ionizing radiation. The contribution of BRCA1 in NHEJ was evaluated in these cells using three different assay systems. First, transfection of a linearized plasmid in which expression of the reporter gene required precise end joining indicated that Brca1 ؊/؊ MEFs display a moderate deficiency when compared with Brca1 ؉/؉ cells. Second, using a retrovirus infection assay dependent on NHEJ, a 5-10-fold reduction in retroviral integration efficiency was observed in Brca1 ؊/؊ MEFs when compared with the Brca1 ؉/؉ MEFs. Third, Brca1 ؊/؊ MEFs exhibited a 50-100-fold deficiency in microhomology-mediated endjoining activity of a defined chromosomal DNA double strand break introduced by a rare cutting endonuclease I-SceI. These results provide evidence that Brca1 has an essential role in microhomology-mediated end joining and suggest a novel molecular basis for its caretaker role in the maintenance of genome integrity.
Background: SUV3 helicase is an essential component of mitochondrial degradosome. Results: Using ... more Background: SUV3 helicase is an essential component of mitochondrial degradosome. Results: Using specific genetic mutants, SUV3 was demonstrated to participate in mtDNA replication, which requires ATPase activity and the C-terminal conserved region. Conclusion: SUV3 plays two distinctive roles in maintaining mtDNA stability and RNA turnover. Significance: This study suggests a direct role of SUV3 in maintaining mtDNA stability, paving a foundation for biochemical investigation. Yeast SUV3 is a nuclear encoded mitochondrial RNA helicase that complexes with an exoribonuclease, DSS1, to function as an RNA degradosome. Inactivation of SUV3 leads to mitochondrial dysfunctions, such as respiratory deficiency; accumulation of aberrant RNA species, including excised group I introns; and loss of mitochondrial DNA (mtDNA). Although intron toxicity has long been speculated to be the major reason for the observed phenotypes, direct evidence to support or refute this theory is lacking. Moreover, it remains unknown whether SUV3 plays a direct role in mtDNA maintenance independently of its degradosome activity. In this paper, we address these questions by employing an inducible knockdown system in Saccharomyces cerevisiae with either normal or intronless mtDNA background. Expressing mutants defective in ATPase (K245A) or RNA binding activities (V272L or ⌬CC, which carries an 8-amino acid deletion at the C-terminal conserved region) resulted in not only respiratory deficiencies but also loss of mtDNA under normal mtDNA background. Surprisingly, V272L, but not other mutants, can rescue the said deficiencies under intronless background. These results provide genetic evidence supporting the notion that the functional requirements of SUV3 for degradosome activity and maintenance of mtDNA stability are separable. Furthermore, V272L mutants and wild-type SUV3 associated with an active mtDNA replication origin and facilitated mtDNA replication, whereas K245A and ⌬CC failed to support mtDNA replication. These results indicate a direct role of SUV3 in maintaining mitochondrial genome stability that is independent of intron turnover but requires the intact ATPase activity and the CC conserved region.
RAD51 recombinase activity plays a critical role for cancer cell proliferation and survival, and ... more RAD51 recombinase activity plays a critical role for cancer cell proliferation and survival, and often contributes to drug-resistance. Abnormally elevated RAD51 function and hyperactive homologous recombination (HR) rates have been found in a panel of cancers, including breast cancer and chronic myeloid leukaemia (CML). Directly targeting RAD51 and attenuating the deregulated RAD51 activity has therefore been proposed as an alternative and supplementary strategy for cancer treatment. Here we show that a newly identified small molecule, IBR2, disrupts RAD51 multimerization, accelerates proteasome-mediated RAD51 protein degradation, reduces ionizing radiation-induced RAD51 foci formation, impairs HR, inhibits cancer cell growth and induces apoptosis. In a murine imatinib-resistant CML model bearing the T315I Bcr-abl mutation, IBR2, but not imatinib, significantly prolonged animal survival. Moreover, IBR2 effectively inhibits the proliferation of CD34 þ progenitor cells from CML patients resistant to known BCR-ABL inhibitors. Therefore, small molecule inhibitors of RAD51 may suggest a novel class of broad-spectrum therapeutics for difficult-to-treat cancers.
Suv3 of Saccharomyces cerevisiae has been classified as a mitochondrial RNA helicase. However, th... more Suv3 of Saccharomyces cerevisiae has been classified as a mitochondrial RNA helicase. However, the helicase domain in both yeast and human SUV3 varies considerably from the typical RNA helicase motifs. To investigate its enzymatic activities, a homogeneously purified preparation of SUV3 is required. Expression of a processed form of human SUV3 carrying an N-terminal deletion of 46 amino acids (SUV3DeltaN46) in a yeast suv3 null mutant, which otherwise fails to grow in a nonfermentable carbon source and forms petite colonies in glucose medium, rescues the null phenotype. Through a five-step chromatographic procedure, an 83 kDa SUV3DeltaN46 protein (SUV3-83) and a partially degraded 70 kDa product (SUV3-70) containing amino acids 68-685 were purified to homogeneity. Single- or double-stranded DNA and RNA stimulated ATPase activity of both proteins. SUV3-70, which retains core catalytic domains, can bind and unwind multiple duplex substrates of RNA and DNA with a 5'-3' directionality over a wide range of pH, while SUV3-83 has helicase activity at only acidic pH. ATP, but not nonhydrolyzable ATP, is essential for the unwinding activity, suggesting the requirement of the energy derived from ATP hydrolysis. Consistent with this notion, suv3 mutants containing alanine (A) or arginine (R) substitutions at the conserved lysine residue in the ATP binding site (K213) lost ATPase activity and also failed to unwind the substrates. Importantly, circular dichroism (CD) spectral analysis showed that SUV3-83, at pH 5.0, adopts a conformation similar to that of SUV3-70, suggesting a conformational change in SUV3-83 is required for its helicase activity. The physiological relevance of the multiple-substrate helicase activity of human SUV3 is discussed.
The 26S proteasome is a multisubunit complex responsible for degradation of ubiquitinated substra... more The 26S proteasome is a multisubunit complex responsible for degradation of ubiquitinated substrates, which plays a critical role in regulating various biological processes. To fully understand the function and regulation of the proteasome complex, an important step is to elucidate its subunit composition and posttranslational modifications. Toward this goal, a new affinity purification strategy has been developed using a derivative of the HB tag for rapid isolation of the human 26S proteasome complex for subsequent proteomic analysis. The purification of the complex is achieved from stable 293 cell lines expressing a HB-tagged proteasome subunit and by high-affinity streptavidin binding with TEV cleavage elution. The complete composition of the 26S proteasome complex, including recently assigned new subunits, is identified by LC-MS/MS. In addition, all known proteasome activator proteins and components involved in the ubiquitin-proteasome degradation pathway are identified. Aside from the subunit composition, the N-terminal modification and phosphorylation of the proteasome subunits have been characterized. Twelve novel phosphorylation sites from eight subunits have been identified, and N-terminal modifications are determined for 25 subunits, 12 of which have not been previously reported in mammals. We also observe different N-terminal processing of subunit Rpn2, which results in identification of two different N-termini of the protein. This work presents the first comprehensive characterization of the human 26S proteasome complex by affinity purification and tandem mass spectrometry. The detailed proteomic profiling obtained here is significant to future studies aiming at a complete understanding of the structure-function relationship of the human 26S proteasome complex.
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Papers by Chi-Fen Chen