We reported that tumor content of prothymosin ␣ (ProT ␣) is a proliferation index of human breast... more We reported that tumor content of prothymosin ␣ (ProT ␣) is a proliferation index of human breast tumors that might be used to identify patients at high risk for distant metastasis (Dominguez et al., Eur J Cancer 1993; 29A:893-7). In that study ProT ␣ concentrations were measured by a RIA; here we present an alternative nonisotopic assay that could be used in a standard clinical laboratory. Main features of the ELISA are: (a) A recombinant fusion protein glutathione S-transferase (GST)-human ProT ␣ was used to coat the microtiter plates; (b) we used a polyclonal antiserum raised in rabbits that detects thymosin ␣ 1 , the NH 2 -terminal fragment of ProT ␣; (c) it is as sensitive as the RIA; (d) it is faster than the RIA. ProT ␣ concentrations in various human tumors (skin, esophagus, colorectal, and breast) as assessed by ELISA were comparable with, although twofold greater than, the values previously estimated by RIA.
Expression of prothymosin a (PTA) has been related to cell proliferation, both normal and patholo... more Expression of prothymosin a (PTA) has been related to cell proliferation, both normal and pathological. PTA has also been proposed to be a target of the c-myc protooncogene. To study PTA mRNA levels during pathological cell growth, and especially the effect of the activation of specific oncogenes on PTA expression, we have studied its expression in tumors that arise in transgenic mice. We found high PTA levels in mammary tumors arising in c-myc, c-neu, and v-ras transgenic mice. Levels of this protein were variable between different tumors, and there is a differential regulation of PTA respect to other putative c-myc target genes, such as Omithine Decarboxylase (ODC). Furthermore, expression of PTA is not absolutely dependent of c-myc expression, as shown by MYC depletion experiments performed with antisense oligonucleotides. We conclude that regulation of PTA in these tumors is complex and depends on more than a single activated oncogene.
Little is known about the TGF-b1 mechanism that promotes thyroid cell growth arrest. We assessed ... more Little is known about the TGF-b1 mechanism that promotes thyroid cell growth arrest. We assessed TGF-b1 eects on Fisher rat thyroid cell line . This allowed us to study TGF-b1 action on thyroid cells in various physiological situations such as actively proliferating cells, resting cells stimulated to proliferate by the action of various mitogens, and resting cells. TGF-b1 arrested proliferating FRTL-5 cells, increasing c-myc mRNA levels and reducing p27-free cyclin D1 protein levels, without aecting either the cellular content of p27 or the cyclin D1-p27 complexes. Moreover, TGF-b1 treatment reduced the activity of cyclin E-CDK2 complexes and, consequently, pRB was found to be hypophosphorylated. TGF-b1 prevented resting cells to enter in the cell cycle when stimulated with growing medium (newborn calf serum plus a mixture of ®ve hormones) but not when TSH (thyroid stimulating hormone) plus IGF-1 (Insulin-like growth factor I) were used as mitogens. Both stimuli increased the levels of cyclins D1, D3 and E but TGF-b1 had a greater eect in decreasing these cyclin levels in growing-medium stimulated cells than in TSH+IGF-1. This suggests that for FRTL-5 cells, the content of these cyclins must exceed a threshold to progress through the cell cycle. TGF-b1 induced apoptosis in quiescent cells, accompanied by a reduction in p27 protein levels and an increase in c-myc expression. Interestingly, TGF-b1-induced variations in prothymosin alpha and c-myc mRNA levels were not correlated. TGF-b1 always promoted an increase of p15 mRNA levels. In summary, our results point to the fact that TGF-b1 could play a physiological role in the control of thyroid growth through the modi®cation of cell cycle regulatory proteins.
The levels of thymosin β4 mRNA were studied throughout the cell cycle of NIH 3T3 cells. In serum ... more The levels of thymosin β4 mRNA were studied throughout the cell cycle of NIH 3T3 cells. In serum deprived, quiescent cells, the levels of thymosin β4 were undetectable; after serum restoration, the cells were induced to proliferate and we found a pronounced increase in thymosin β4 mRNA levels at the G1/S transition. Thymosin β4 mRNA was induced even in the presence of cycloheximide. On the other hand, cycling cells that were synchronized at different stages of the cycle by means of mitotic shake-off after nocodazole arrest or a double thymidine block did not show any variation in the levels of thymosin β4 mRNA when they progressed synchronously through the cycle. In conclusion, the present data indicate that the thymosin β4 gene is regulated by cell proliferation but it is not a cell cycle-regulated gene. Finally, we studied thymosin β4 mRNA stability by inhibiting thymosin β4 gene transcription with actinomycin D. Our results suggest that thymosin β4 mRNA has a pronounced stability, a fact that might be relevant to account for the presence of thymosin β4 in enucleated cells like platelets.
ABSTRACT Using flow cytometry we observed the effects that different hormonal treatments had on t... more ABSTRACT Using flow cytometry we observed the effects that different hormonal treatments had on the progression of rat thyroid (FRTL-5) cells through the cell cycle. The absence of hormones or the addition of TSH (6 mU/ml) did not induce DNA synthesis; however, the addition of IGF-I (30 ng/ml) promoted cell proliferation. The number of cells recruited by IGF-I was lower than when IGF-I and TSH were used. We therefore concluded that we had a model with three different types of cells: (1) quiescent cells, cells cultured in the absence of hormones, considered to be G0-arrested cells, (2) competent cells, TSH-treated cells that did not proliferate (being arrested in a cycle phase different from G0) and (3) actively proliferating cells, cells treated with TSH plus IGF-I. Prothymosin alpha (PTA) mRNA levels were almost undetectable in cells cultured without hormones at all times studied, i.e. 8, 14 and 24 h. On the contrary, TSH and/or IGF-I greatly increased PTA mRNA. These data indicate that G0-arrested quiescent cells do not express PTA mRNA and that PTA mRNA is induced when FRTL-5 cells are committed to proliferate by the addition of TSH, in spite of being arrested by the lack of IGF-I. We therefore conclude that PTA mRNA expression may be an event that is necessary for cells to proliferate, but that it is not sufficient for the promotion of cell progression through the cell cycle.
In 71 patents with classic invasive ductal carcinomas, levels of prothymosin alpha (PT~), as assa... more In 71 patents with classic invasive ductal carcinomas, levels of prothymosin alpha (PT~), as assayed by a radioimmunoassay that detects thymosin alpha 1 (the NHe-terminal fragment of PTo0, were significantly greater in tumour samples than in normal breast tissue. PTot levels were correlated with (a) the number of positive axillary lymph nodes (rs = 0.5384, P < 0.01), and (b) the percentage of tumour ceils in the S or G2/M phase as assessed by flow cytometry (rs = 0.5027, P < 0.01). Since the beginning of this study in 1989, 21 patients have presented distant metastases, all of whom were previously shown to have tumour PTo~ levels greater than 124 ng of thymosin alpha 1/mg protein. The present report indicates that PTot might be used to identify breast cancer patients at high risk for distant metastases.
Mutations in CCM3/PDCD10 result in cerebral cavernous malformations (CCMs), a major cause of cere... more Mutations in CCM3/PDCD10 result in cerebral cavernous malformations (CCMs), a major cause of cerebral hemorrhage. Despite intense interest in CCMs, very little is known about the function of CCM3. Here, we report that CCM3 is located on the Golgi apparatus, forming a complex with proteins of the germinal center kinase III (GCKIII) family and GM130, a Golgi-resident protein. Cells depleted of CCM3 show a disassembled Golgi apparatus. Furthermore, in wound-healing assays, CCM3-depleted cells cannot reorient the Golgi and centrosome properly, and demonstrate impaired migration. Golgi disassembly after either depletion of CCM3 or dissociation of CCM3 from the GM130-GCKIII complex is the result of destabilization of GCKIII proteins and dephosphorylation of their substrate, 14-3-3. Significantly, the phenotype induced by CCM3 depletion can be reverted by expression of wild-type CCM3, but not by diseaseassociated mutants. Our findings suggest that Golgi dysfunction and the ensuing abnormalities of cell orientation and migration resulting from CCM3 mutations contribute to CCM pathogenesis.
Prothymosin alpha (PTA) mRNA and histone H4 (H4) mRNA levels were studied in various experimental... more Prothymosin alpha (PTA) mRNA and histone H4 (H4) mRNA levels were studied in various experimental conditions that affected GH1 pituitary tumor cell proliferation. Cell proliferation and progression through the cell cycle was assessed by counting cells, 3H-thymidine incorporation and flow cytometry. PTA mRNA levels were decreased in a time-dependent fashion following serum deprivation; when the cells were induced to grow by serum refeeding, PTA mRNA expression was greatly stimulated. Interestingly, after caprylic acid treatment (2.5 mM for 24 h) that arrested cells in the G0/G1 phase of the cell cycle, PTA mRNA and H4 mRNA levels were almost undetectable; conversely, following caprylic acid withdrawal, PTA mRNA and H4 mRNA expression were greatly stimulated. Furthermore, cells cultured in T3-deprived serum, which was found to decrease GH1 cell proliferation, had low levels of PTA and H4 mRNAs. This effect was reversed by the addition of nanomolar concentrations of T3 to the culture. On the other hand, IGF-1 addition to the culture did not substantially modify PTA mRNA levels. The present data clearly indicate that PTA mRNA expression is tied to the proliferating activity of GH1 cells and, thus, could be used as a marker of the action that various agents have on GH1 cell proliferation.
GH-releasing hormone (GHRH) can induce proliferation of somatotroph cells. The pathway involving ... more GH-releasing hormone (GHRH) can induce proliferation of somatotroph cells. The pathway involving adenylyl cyclase/cAMP/protein kinase A pathway in its target cells seems to be important for this action, or at least it is deregulated in some somatotroph pituitary adenomas. We studied in this work whether GHRH can also stimulate mitogen-activated protein (MAP) kinase. GHRH can activate MAP kinase both in pituitary cells and in a cell line overexpressing the GHRH receptor. Although both protein kinase A and protein kinase C could activate MAP kinase in the CHO cell line studied, neither protein kinase A nor protein kinase C appears to be required for GHRH activation of MAP kinase in this system. However, seques-tration of the ␥-subunits of the G protein coupled to the receptor inhibits MAP kinase activation mediated by GHRH. This pathway also involves p21 ras and a phosphatidylinositol 3-kinase, probably phosphatidylinositol 3-kinase-␥. Despite the involvement of p21 ras , the protein kinase Raf-1 is not hyperphosphorylated in response to GHRH, contrary to what usually occurs when the Ras-Raf-MAP kinase pathway is activated. In summary, this work describes for the first time the activation of MAP kinase by GHRH and outlines a path for this activation that is different from the cAMP-dependent mechanism that has been traditionally described as mediating the mitogenic actions of GHRH. (Endocrinology 141
Despite intensive study, the mechanisms regulating activation of mTOR and the consequences of tha... more Despite intensive study, the mechanisms regulating activation of mTOR and the consequences of that activation in the ischemic heart remain unclear. This is particularly true for the setting of ischemia/reperfusion (
H Stubdal, J Zalvide and J A DeCaprio proteins p130 and p107. phosphorylation state of the RB-rel... more H Stubdal, J Zalvide and J A DeCaprio proteins p130 and p107. phosphorylation state of the RB-related Simian virus 40 large T antigen alters the http://jvi.asm.org/content/70/5/2781 Updated information and services can be found at: These include: CONTENT ALERTS more» cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new articles http://journals.asm.org/site/misc/reprints.xhtml Information about commercial reprint orders: http://journals.asm.org/site/subscriptions/ To subscribe to to another ASM Journal go to: on December 12, 2013 by guest http://jvi.asm.org/ Downloaded from on December 12, 2013 by guest p130 and p107 are nuclear phosphoproteins related to the retinoblastoma gene product (pRb). pRb, p107, and p130 each undergo cell cycle-dependent phosphorylation, form complexes with the E2F family of transcription factors, and associate with oncoproteins of DNA tumor viruses, including simian virus 40 (SV40) large T antigen (TAg) and adenovirus E1A protein. The results of recent studies with mouse embryo fibroblasts (MEFs) lacking the retinoblastoma gene (Rb-1) have suggested that p130 and p107 may be important targets for SV40 large TAg-mediated transformation (J. B. Christensen and M. J. Imperiale, J. Virol. 65:3945-3948, 1995; J. Zalvide and J. A. DeCaprio, Mol. Cell. Biol . In this report, we demonstrate that the expression of TAg affects the phosphorylation state of p130 and p107. In cells expressing wild-type TAg, only un(der)phosphorylated p130 and p107 were detected. To determine the domains within TAg that contribute to this effect on the phosphorylation of p130, we performed transient expression assays. While transiently expressed p130 was apparently phosphorylated normally, only un(der)phosphorylated p130 was detected when p130 was coexpressed with TAg. Using this assay, we found that the first 147 amino acids of TAg were sufficient to alter the phosphorylation state of p130. Within this region, the LXCXE domain of TAg, required for binding to the retinoblastoma family of proteins, was necessary but not sufficient to affect p130 phosphorylation. Residues within the first 82 amino acids of TAg were also required. TAg with mutations in the N terminus retained the ability to efficiently associate with p130 but did not affect its phosphorylation state. This demonstrates that the effect of SV40 TAg on p130 is not simply the result of binding and suggests that TAg has a novel effect on p130 and p107 that differs from its effect on pRb.
Inactivation of the retinoblastoma tumor suppressor protein (pRB) contributes to tumorigenesis in... more Inactivation of the retinoblastoma tumor suppressor protein (pRB) contributes to tumorigenesis in a wide variety of cancers. In contrast, the role of the two pRB-related proteins, p130 and p107, in oncogenic transformation is unclear. The LXCXE domain of simian virus 40 large T antigen (TAg) specifically binds to pRB, p107, and p130. We have previously shown that the N terminus and the LXCXE domain of TAg cooperate to alter the phosphorylation state of p130 and p107. Here, we demonstrate that TAg promotes the degradation of p130 and that the N terminus of TAg is required for this activity. The N terminus of TAg has homology to the J domain of the DnaJ family of molecular chaperone proteins. Mutants with mutations in the J-domain homology region of TAg are defective for altering p130 and p107 phosphorylation and for p130 degradation. A heterologous J-domain from a human DnaJ protein can functionally substitute for the N terminus of TAg in the effect on p107 and p130 phosphorylation and p130 stability. We further demonstrate that the J-domain homology region of TAg confers a growth advantage to wild-type mouse embryo fibroblasts (MEFs) but is dispensable in the case of MEFs lacking both p130 and p107. This indicates that p107 and p130 have overlapping growth-suppressing activities whose inactivation is mediated by the J domain of TAg.
Access the most recent version at doi: 1997 11: 1098-1110 Genes Dev. K S Campbell, K P Mullane, I... more Access the most recent version at doi: 1997 11: 1098-1110 Genes Dev. K S Campbell, K P Mullane, I A Aksoy, et al. efficient viral DNA replication. DnaJ/hsp40 chaperone domain of SV40 large T antigen promotes References http://genesdev.cshlp.org/content/11/9/1098#related-urls Article cited in: http://genesdev.cshlp.org/content/11/9/1098.refs.html The amino-terminal domain of SV40 large tumor antigen (TAg) is required for efficient viral DNA replication. However, the biochemical activity associated with this domain has remained obscure. We show here that the amino-terminal domain of TAg shares functional homology with the J-domain of DnaJ/hsp40 molecular chaperones. DnaJ proteins function as cofactors by regulating the activity of a member of the 70-kD heat shock protein family.
Access the most recent version at doi: 1997 11: 1098-1110 Genes Dev. K S Campbell, K P Mullane, I... more Access the most recent version at doi: 1997 11: 1098-1110 Genes Dev. K S Campbell, K P Mullane, I A Aksoy, et al. efficient viral DNA replication. DnaJ/hsp40 chaperone domain of SV40 large T antigen promotes References http://genesdev.cshlp.org/content/11/9/1098#related-urls Article cited in: http://genesdev.cshlp.org/content/11/9/1098.refs.html The amino-terminal domain of SV40 large tumor antigen (TAg) is required for efficient viral DNA replication. However, the biochemical activity associated with this domain has remained obscure. We show here that the amino-terminal domain of TAg shares functional homology with the J-domain of DnaJ/hsp40 molecular chaperones. DnaJ proteins function as cofactors by regulating the activity of a member of the 70-kD heat shock protein family.
Proceedings of The National Academy of Sciences, 1996
Cyclin E is an important regulator of cell cycle progression that together with cyclin-dependent ... more Cyclin E is an important regulator of cell cycle progression that together with cyclin-dependent kinase (cdk) 2 is crucial for the G 1 ͞S transition during the mammalian cell cycle. Previously, we showed that severe overexpression of cyclin E protein in tumor cells and tissues results in the appearance of lower molecular weight isoforms of cyclin E, which together with cdk2 can form a kinase complex active throughout the cell cycle. In this study, we report that one of the substrates of this constitutively active cyclin E͞cdk2 complex is retinoblastoma susceptibility gene product (pRb) in populations of breast cancer cells and tissues that also overexpress p16. In these tumor cells and tissues, we show that the expression of p16 and pRb is not mutually exclusive. Overexpression of p16 in these cells results in sequestering of cdk4 and cdk6, rendering cyclin D1͞cdk complexes inactive. However, pRb appears to be phosphorylated throughout the cell cycle following an initial lag, revealing a time course similar to phosphorylation of glutathione S-transferase retinoblastoma by cyclin E immunoprecipitates prepared from these synchronized cells. Hence, cyclin E kinase complexes can function redundantly and replace the loss of cyclin Ddependent kinase complexes that functionally inactivate pRb. In addition, the constitutively overexpressed cyclin E is also the predominant cyclin found in p107͞E2F complexes throughout the tumor, but not the normal, cell cycle. These observations suggest that overexpression of cyclin E in tumor cells, which also overexpress p16, can bypass the cyclin D͞cdk4-cdk6͞p16͞pRb feedback loop, providing yet another mechanism by which tumors can gain a growth advantage.
Transformation by simian virus 40 large T antigen (TAg) is dependent on the inactivation of cellu... more Transformation by simian virus 40 large T antigen (TAg) is dependent on the inactivation of cellular tumor suppressors. Transformation minimally requires the following three domains: (i) a C-terminal domain that mediates binding to p53; (ii) the LXCXE domain (residues 103 to 107), necessary for binding to the retinoblastoma tumor suppressor protein, pRB, and the related p107 and p130; and (iii) an N-terminal domain that is homologous to the J domain of DnaJ molecular chaperone proteins. We have previously demonstrated that the N-terminal J domain of TAg affects the RB-related proteins by perturbing the phosphorylation status of p107 and p130 and promoting the degradation of p130 and that this domain is required for transformation of cells that express either p107 or p130. In this work, we demonstrate that the J domain of TAg is required to inactivate the ability of each member of the pRB family to induce a G 1 arrest in Saos-2 cells. Furthermore, the J domain is required to override the repression of E2F activity mediated by p130 and pRB and to disrupt p130-E2F DNA binding complexes. These results imply that while the LXCXE domain serves as a binding site for the RB-related proteins, the J domain plays an important role in inactivating their function.
We reported that tumor content of prothymosin ␣ (ProT ␣) is a proliferation index of human breast... more We reported that tumor content of prothymosin ␣ (ProT ␣) is a proliferation index of human breast tumors that might be used to identify patients at high risk for distant metastasis (Dominguez et al., Eur J Cancer 1993; 29A:893-7). In that study ProT ␣ concentrations were measured by a RIA; here we present an alternative nonisotopic assay that could be used in a standard clinical laboratory. Main features of the ELISA are: (a) A recombinant fusion protein glutathione S-transferase (GST)-human ProT ␣ was used to coat the microtiter plates; (b) we used a polyclonal antiserum raised in rabbits that detects thymosin ␣ 1 , the NH 2 -terminal fragment of ProT ␣; (c) it is as sensitive as the RIA; (d) it is faster than the RIA. ProT ␣ concentrations in various human tumors (skin, esophagus, colorectal, and breast) as assessed by ELISA were comparable with, although twofold greater than, the values previously estimated by RIA.
Expression of prothymosin a (PTA) has been related to cell proliferation, both normal and patholo... more Expression of prothymosin a (PTA) has been related to cell proliferation, both normal and pathological. PTA has also been proposed to be a target of the c-myc protooncogene. To study PTA mRNA levels during pathological cell growth, and especially the effect of the activation of specific oncogenes on PTA expression, we have studied its expression in tumors that arise in transgenic mice. We found high PTA levels in mammary tumors arising in c-myc, c-neu, and v-ras transgenic mice. Levels of this protein were variable between different tumors, and there is a differential regulation of PTA respect to other putative c-myc target genes, such as Omithine Decarboxylase (ODC). Furthermore, expression of PTA is not absolutely dependent of c-myc expression, as shown by MYC depletion experiments performed with antisense oligonucleotides. We conclude that regulation of PTA in these tumors is complex and depends on more than a single activated oncogene.
Little is known about the TGF-b1 mechanism that promotes thyroid cell growth arrest. We assessed ... more Little is known about the TGF-b1 mechanism that promotes thyroid cell growth arrest. We assessed TGF-b1 eects on Fisher rat thyroid cell line . This allowed us to study TGF-b1 action on thyroid cells in various physiological situations such as actively proliferating cells, resting cells stimulated to proliferate by the action of various mitogens, and resting cells. TGF-b1 arrested proliferating FRTL-5 cells, increasing c-myc mRNA levels and reducing p27-free cyclin D1 protein levels, without aecting either the cellular content of p27 or the cyclin D1-p27 complexes. Moreover, TGF-b1 treatment reduced the activity of cyclin E-CDK2 complexes and, consequently, pRB was found to be hypophosphorylated. TGF-b1 prevented resting cells to enter in the cell cycle when stimulated with growing medium (newborn calf serum plus a mixture of ®ve hormones) but not when TSH (thyroid stimulating hormone) plus IGF-1 (Insulin-like growth factor I) were used as mitogens. Both stimuli increased the levels of cyclins D1, D3 and E but TGF-b1 had a greater eect in decreasing these cyclin levels in growing-medium stimulated cells than in TSH+IGF-1. This suggests that for FRTL-5 cells, the content of these cyclins must exceed a threshold to progress through the cell cycle. TGF-b1 induced apoptosis in quiescent cells, accompanied by a reduction in p27 protein levels and an increase in c-myc expression. Interestingly, TGF-b1-induced variations in prothymosin alpha and c-myc mRNA levels were not correlated. TGF-b1 always promoted an increase of p15 mRNA levels. In summary, our results point to the fact that TGF-b1 could play a physiological role in the control of thyroid growth through the modi®cation of cell cycle regulatory proteins.
The levels of thymosin β4 mRNA were studied throughout the cell cycle of NIH 3T3 cells. In serum ... more The levels of thymosin β4 mRNA were studied throughout the cell cycle of NIH 3T3 cells. In serum deprived, quiescent cells, the levels of thymosin β4 were undetectable; after serum restoration, the cells were induced to proliferate and we found a pronounced increase in thymosin β4 mRNA levels at the G1/S transition. Thymosin β4 mRNA was induced even in the presence of cycloheximide. On the other hand, cycling cells that were synchronized at different stages of the cycle by means of mitotic shake-off after nocodazole arrest or a double thymidine block did not show any variation in the levels of thymosin β4 mRNA when they progressed synchronously through the cycle. In conclusion, the present data indicate that the thymosin β4 gene is regulated by cell proliferation but it is not a cell cycle-regulated gene. Finally, we studied thymosin β4 mRNA stability by inhibiting thymosin β4 gene transcription with actinomycin D. Our results suggest that thymosin β4 mRNA has a pronounced stability, a fact that might be relevant to account for the presence of thymosin β4 in enucleated cells like platelets.
ABSTRACT Using flow cytometry we observed the effects that different hormonal treatments had on t... more ABSTRACT Using flow cytometry we observed the effects that different hormonal treatments had on the progression of rat thyroid (FRTL-5) cells through the cell cycle. The absence of hormones or the addition of TSH (6 mU/ml) did not induce DNA synthesis; however, the addition of IGF-I (30 ng/ml) promoted cell proliferation. The number of cells recruited by IGF-I was lower than when IGF-I and TSH were used. We therefore concluded that we had a model with three different types of cells: (1) quiescent cells, cells cultured in the absence of hormones, considered to be G0-arrested cells, (2) competent cells, TSH-treated cells that did not proliferate (being arrested in a cycle phase different from G0) and (3) actively proliferating cells, cells treated with TSH plus IGF-I. Prothymosin alpha (PTA) mRNA levels were almost undetectable in cells cultured without hormones at all times studied, i.e. 8, 14 and 24 h. On the contrary, TSH and/or IGF-I greatly increased PTA mRNA. These data indicate that G0-arrested quiescent cells do not express PTA mRNA and that PTA mRNA is induced when FRTL-5 cells are committed to proliferate by the addition of TSH, in spite of being arrested by the lack of IGF-I. We therefore conclude that PTA mRNA expression may be an event that is necessary for cells to proliferate, but that it is not sufficient for the promotion of cell progression through the cell cycle.
In 71 patents with classic invasive ductal carcinomas, levels of prothymosin alpha (PT~), as assa... more In 71 patents with classic invasive ductal carcinomas, levels of prothymosin alpha (PT~), as assayed by a radioimmunoassay that detects thymosin alpha 1 (the NHe-terminal fragment of PTo0, were significantly greater in tumour samples than in normal breast tissue. PTot levels were correlated with (a) the number of positive axillary lymph nodes (rs = 0.5384, P < 0.01), and (b) the percentage of tumour ceils in the S or G2/M phase as assessed by flow cytometry (rs = 0.5027, P < 0.01). Since the beginning of this study in 1989, 21 patients have presented distant metastases, all of whom were previously shown to have tumour PTo~ levels greater than 124 ng of thymosin alpha 1/mg protein. The present report indicates that PTot might be used to identify breast cancer patients at high risk for distant metastases.
Mutations in CCM3/PDCD10 result in cerebral cavernous malformations (CCMs), a major cause of cere... more Mutations in CCM3/PDCD10 result in cerebral cavernous malformations (CCMs), a major cause of cerebral hemorrhage. Despite intense interest in CCMs, very little is known about the function of CCM3. Here, we report that CCM3 is located on the Golgi apparatus, forming a complex with proteins of the germinal center kinase III (GCKIII) family and GM130, a Golgi-resident protein. Cells depleted of CCM3 show a disassembled Golgi apparatus. Furthermore, in wound-healing assays, CCM3-depleted cells cannot reorient the Golgi and centrosome properly, and demonstrate impaired migration. Golgi disassembly after either depletion of CCM3 or dissociation of CCM3 from the GM130-GCKIII complex is the result of destabilization of GCKIII proteins and dephosphorylation of their substrate, 14-3-3. Significantly, the phenotype induced by CCM3 depletion can be reverted by expression of wild-type CCM3, but not by diseaseassociated mutants. Our findings suggest that Golgi dysfunction and the ensuing abnormalities of cell orientation and migration resulting from CCM3 mutations contribute to CCM pathogenesis.
Prothymosin alpha (PTA) mRNA and histone H4 (H4) mRNA levels were studied in various experimental... more Prothymosin alpha (PTA) mRNA and histone H4 (H4) mRNA levels were studied in various experimental conditions that affected GH1 pituitary tumor cell proliferation. Cell proliferation and progression through the cell cycle was assessed by counting cells, 3H-thymidine incorporation and flow cytometry. PTA mRNA levels were decreased in a time-dependent fashion following serum deprivation; when the cells were induced to grow by serum refeeding, PTA mRNA expression was greatly stimulated. Interestingly, after caprylic acid treatment (2.5 mM for 24 h) that arrested cells in the G0/G1 phase of the cell cycle, PTA mRNA and H4 mRNA levels were almost undetectable; conversely, following caprylic acid withdrawal, PTA mRNA and H4 mRNA expression were greatly stimulated. Furthermore, cells cultured in T3-deprived serum, which was found to decrease GH1 cell proliferation, had low levels of PTA and H4 mRNAs. This effect was reversed by the addition of nanomolar concentrations of T3 to the culture. On the other hand, IGF-1 addition to the culture did not substantially modify PTA mRNA levels. The present data clearly indicate that PTA mRNA expression is tied to the proliferating activity of GH1 cells and, thus, could be used as a marker of the action that various agents have on GH1 cell proliferation.
GH-releasing hormone (GHRH) can induce proliferation of somatotroph cells. The pathway involving ... more GH-releasing hormone (GHRH) can induce proliferation of somatotroph cells. The pathway involving adenylyl cyclase/cAMP/protein kinase A pathway in its target cells seems to be important for this action, or at least it is deregulated in some somatotroph pituitary adenomas. We studied in this work whether GHRH can also stimulate mitogen-activated protein (MAP) kinase. GHRH can activate MAP kinase both in pituitary cells and in a cell line overexpressing the GHRH receptor. Although both protein kinase A and protein kinase C could activate MAP kinase in the CHO cell line studied, neither protein kinase A nor protein kinase C appears to be required for GHRH activation of MAP kinase in this system. However, seques-tration of the ␥-subunits of the G protein coupled to the receptor inhibits MAP kinase activation mediated by GHRH. This pathway also involves p21 ras and a phosphatidylinositol 3-kinase, probably phosphatidylinositol 3-kinase-␥. Despite the involvement of p21 ras , the protein kinase Raf-1 is not hyperphosphorylated in response to GHRH, contrary to what usually occurs when the Ras-Raf-MAP kinase pathway is activated. In summary, this work describes for the first time the activation of MAP kinase by GHRH and outlines a path for this activation that is different from the cAMP-dependent mechanism that has been traditionally described as mediating the mitogenic actions of GHRH. (Endocrinology 141
Despite intensive study, the mechanisms regulating activation of mTOR and the consequences of tha... more Despite intensive study, the mechanisms regulating activation of mTOR and the consequences of that activation in the ischemic heart remain unclear. This is particularly true for the setting of ischemia/reperfusion (
H Stubdal, J Zalvide and J A DeCaprio proteins p130 and p107. phosphorylation state of the RB-rel... more H Stubdal, J Zalvide and J A DeCaprio proteins p130 and p107. phosphorylation state of the RB-related Simian virus 40 large T antigen alters the http://jvi.asm.org/content/70/5/2781 Updated information and services can be found at: These include: CONTENT ALERTS more» cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new articles http://journals.asm.org/site/misc/reprints.xhtml Information about commercial reprint orders: http://journals.asm.org/site/subscriptions/ To subscribe to to another ASM Journal go to: on December 12, 2013 by guest http://jvi.asm.org/ Downloaded from on December 12, 2013 by guest p130 and p107 are nuclear phosphoproteins related to the retinoblastoma gene product (pRb). pRb, p107, and p130 each undergo cell cycle-dependent phosphorylation, form complexes with the E2F family of transcription factors, and associate with oncoproteins of DNA tumor viruses, including simian virus 40 (SV40) large T antigen (TAg) and adenovirus E1A protein. The results of recent studies with mouse embryo fibroblasts (MEFs) lacking the retinoblastoma gene (Rb-1) have suggested that p130 and p107 may be important targets for SV40 large TAg-mediated transformation (J. B. Christensen and M. J. Imperiale, J. Virol. 65:3945-3948, 1995; J. Zalvide and J. A. DeCaprio, Mol. Cell. Biol . In this report, we demonstrate that the expression of TAg affects the phosphorylation state of p130 and p107. In cells expressing wild-type TAg, only un(der)phosphorylated p130 and p107 were detected. To determine the domains within TAg that contribute to this effect on the phosphorylation of p130, we performed transient expression assays. While transiently expressed p130 was apparently phosphorylated normally, only un(der)phosphorylated p130 was detected when p130 was coexpressed with TAg. Using this assay, we found that the first 147 amino acids of TAg were sufficient to alter the phosphorylation state of p130. Within this region, the LXCXE domain of TAg, required for binding to the retinoblastoma family of proteins, was necessary but not sufficient to affect p130 phosphorylation. Residues within the first 82 amino acids of TAg were also required. TAg with mutations in the N terminus retained the ability to efficiently associate with p130 but did not affect its phosphorylation state. This demonstrates that the effect of SV40 TAg on p130 is not simply the result of binding and suggests that TAg has a novel effect on p130 and p107 that differs from its effect on pRb.
Inactivation of the retinoblastoma tumor suppressor protein (pRB) contributes to tumorigenesis in... more Inactivation of the retinoblastoma tumor suppressor protein (pRB) contributes to tumorigenesis in a wide variety of cancers. In contrast, the role of the two pRB-related proteins, p130 and p107, in oncogenic transformation is unclear. The LXCXE domain of simian virus 40 large T antigen (TAg) specifically binds to pRB, p107, and p130. We have previously shown that the N terminus and the LXCXE domain of TAg cooperate to alter the phosphorylation state of p130 and p107. Here, we demonstrate that TAg promotes the degradation of p130 and that the N terminus of TAg is required for this activity. The N terminus of TAg has homology to the J domain of the DnaJ family of molecular chaperone proteins. Mutants with mutations in the J-domain homology region of TAg are defective for altering p130 and p107 phosphorylation and for p130 degradation. A heterologous J-domain from a human DnaJ protein can functionally substitute for the N terminus of TAg in the effect on p107 and p130 phosphorylation and p130 stability. We further demonstrate that the J-domain homology region of TAg confers a growth advantage to wild-type mouse embryo fibroblasts (MEFs) but is dispensable in the case of MEFs lacking both p130 and p107. This indicates that p107 and p130 have overlapping growth-suppressing activities whose inactivation is mediated by the J domain of TAg.
Access the most recent version at doi: 1997 11: 1098-1110 Genes Dev. K S Campbell, K P Mullane, I... more Access the most recent version at doi: 1997 11: 1098-1110 Genes Dev. K S Campbell, K P Mullane, I A Aksoy, et al. efficient viral DNA replication. DnaJ/hsp40 chaperone domain of SV40 large T antigen promotes References http://genesdev.cshlp.org/content/11/9/1098#related-urls Article cited in: http://genesdev.cshlp.org/content/11/9/1098.refs.html The amino-terminal domain of SV40 large tumor antigen (TAg) is required for efficient viral DNA replication. However, the biochemical activity associated with this domain has remained obscure. We show here that the amino-terminal domain of TAg shares functional homology with the J-domain of DnaJ/hsp40 molecular chaperones. DnaJ proteins function as cofactors by regulating the activity of a member of the 70-kD heat shock protein family.
Access the most recent version at doi: 1997 11: 1098-1110 Genes Dev. K S Campbell, K P Mullane, I... more Access the most recent version at doi: 1997 11: 1098-1110 Genes Dev. K S Campbell, K P Mullane, I A Aksoy, et al. efficient viral DNA replication. DnaJ/hsp40 chaperone domain of SV40 large T antigen promotes References http://genesdev.cshlp.org/content/11/9/1098#related-urls Article cited in: http://genesdev.cshlp.org/content/11/9/1098.refs.html The amino-terminal domain of SV40 large tumor antigen (TAg) is required for efficient viral DNA replication. However, the biochemical activity associated with this domain has remained obscure. We show here that the amino-terminal domain of TAg shares functional homology with the J-domain of DnaJ/hsp40 molecular chaperones. DnaJ proteins function as cofactors by regulating the activity of a member of the 70-kD heat shock protein family.
Proceedings of The National Academy of Sciences, 1996
Cyclin E is an important regulator of cell cycle progression that together with cyclin-dependent ... more Cyclin E is an important regulator of cell cycle progression that together with cyclin-dependent kinase (cdk) 2 is crucial for the G 1 ͞S transition during the mammalian cell cycle. Previously, we showed that severe overexpression of cyclin E protein in tumor cells and tissues results in the appearance of lower molecular weight isoforms of cyclin E, which together with cdk2 can form a kinase complex active throughout the cell cycle. In this study, we report that one of the substrates of this constitutively active cyclin E͞cdk2 complex is retinoblastoma susceptibility gene product (pRb) in populations of breast cancer cells and tissues that also overexpress p16. In these tumor cells and tissues, we show that the expression of p16 and pRb is not mutually exclusive. Overexpression of p16 in these cells results in sequestering of cdk4 and cdk6, rendering cyclin D1͞cdk complexes inactive. However, pRb appears to be phosphorylated throughout the cell cycle following an initial lag, revealing a time course similar to phosphorylation of glutathione S-transferase retinoblastoma by cyclin E immunoprecipitates prepared from these synchronized cells. Hence, cyclin E kinase complexes can function redundantly and replace the loss of cyclin Ddependent kinase complexes that functionally inactivate pRb. In addition, the constitutively overexpressed cyclin E is also the predominant cyclin found in p107͞E2F complexes throughout the tumor, but not the normal, cell cycle. These observations suggest that overexpression of cyclin E in tumor cells, which also overexpress p16, can bypass the cyclin D͞cdk4-cdk6͞p16͞pRb feedback loop, providing yet another mechanism by which tumors can gain a growth advantage.
Transformation by simian virus 40 large T antigen (TAg) is dependent on the inactivation of cellu... more Transformation by simian virus 40 large T antigen (TAg) is dependent on the inactivation of cellular tumor suppressors. Transformation minimally requires the following three domains: (i) a C-terminal domain that mediates binding to p53; (ii) the LXCXE domain (residues 103 to 107), necessary for binding to the retinoblastoma tumor suppressor protein, pRB, and the related p107 and p130; and (iii) an N-terminal domain that is homologous to the J domain of DnaJ molecular chaperone proteins. We have previously demonstrated that the N-terminal J domain of TAg affects the RB-related proteins by perturbing the phosphorylation status of p107 and p130 and promoting the degradation of p130 and that this domain is required for transformation of cells that express either p107 or p130. In this work, we demonstrate that the J domain of TAg is required to inactivate the ability of each member of the pRB family to induce a G 1 arrest in Saos-2 cells. Furthermore, the J domain is required to override the repression of E2F activity mediated by p130 and pRB and to disrupt p130-E2F DNA binding complexes. These results imply that while the LXCXE domain serves as a binding site for the RB-related proteins, the J domain plays an important role in inactivating their function.
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