scholarly journals Down-regulation of actin genes precedes microfilament network disruption and actin cleavage during p53-mediated apoptosis

1997 ◽  
Vol 110 (4) ◽  
pp. 489-495 ◽  
Author(s):  
I. Guenal ◽  
Y. Risler ◽  
B. Mignotte
Keyword(s):  
Steady State ◽  
Interleukin 1 ◽  
Simian Virus 40 ◽  
State Level ◽  
Simian Virus ◽  
T Antigen ◽  
Mrna Levels ◽  
Down Regulation ◽  
Actin Genes ◽  
Steady State Levels

Inactivation of Simian Virus 40 large T antigen, in cells immortalized with conditional mutants, leads to activation of p53 and apoptosis. We used the mRNA differential display method to identify genes differentially expressed during this process. We found that steady-state levels of mRNA for cytoplasmic actins decreased early during apoptosis. We also showed that, although the steady-state level of the corresponding proteins is not profoundly affected, they are substrates for an interleukin 1-beta converting enzyme (ICE)-like protease activated during the process. However, only a very small fraction of actin is proteolysed during the early stages of apoptosis. The microfilament network is affected and non polymerized actin accumulates in apoptotic bodies after the decrease of mRNA levels, but before a significant amount of actin is cleaved. This suggests that down-regulation of actin genes may be involved in microfilament rearrangements during p53-mediated apoptosis.

scholarly journals Induction of cellular thymidine kinase occurs at the mRNA level.

1985 ◽  
Vol 5 (6) ◽  
pp. 1490-1497 ◽  
Author(s):  
P Stuart ◽  
M Ito ◽  
C Stewart ◽  
S E Conrad
Keyword(s):  
Steady State ◽  
Thymidine Kinase ◽  
Cdna Clone ◽  
Blot Hybridization ◽  
Mrna Level ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Chromosomal Dna ◽  
Infected Cells ◽  
Steady State Levels

The thymidine kinase (TK) gene has been isolated from human genomic DNA. The gene was passaged twice by transfection of LTK- cells with human chromosomal DNA, and genomic libraries were made in lambda Charon 30 from a second-round TK+ transformant. When the library was screened with a human Alu probe, seven overlapping lambda clones from the human TK locus were obtained. None of the seven contained a functional TK gene as judged by transfection analysis, but several combinations of clones gave rise to TK+ colonies when cotransfected into TK- cells. A functional cDNA clone encoding the human TK gene has also been isolated. Using this cDNA clone as a probe in restriction enzyme/blot hybridization analyses, we have mapped the coding sequences and direction of transcription of the gene. We have also used a single-copy subclone from within the coding region to monitor steady-state levels of TK mRNA in serum-stimulated and simian virus 40-infected simian CV1 tissue culture cells. Our results indicate that the previously reported increase in TK enzyme levels seen after either treatment is paralleled by an equivalent increase in the steady-state levels of TK mRNA. In the case of simian virus 40-infected cells, the induction was delayed by 8 to 12 h, which is the length of time after infection required for early viral protein synthesis. In both cases, induction of TK mRNA coincides with the onset of DNA synthesis, but virally infected cells ultimately accumulate more TK mRNA than do serum-stimulated cells.

scholarly journals Efficiency of utilization of the simian virus 40 late polyadenylation site: effects of upstream sequences.

1989 ◽  
Vol 9 (10) ◽  
pp. 4248-4258 ◽  
Author(s):  
S Carswell ◽  
J C Alwine
Keyword(s):  
Steady State ◽  
Rna Stability ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Polyadenylation Signal ◽  
Polyadenylation Site ◽  
Expression Vectors ◽  
Mrna Levels ◽  
The Difference ◽  
Polyadenylation Signals

The late polyadenylation signal of simian virus 40 functions with greater efficiency than the early polyadenylation signal, in turn affecting steady-state mRNA levels. Two chloramphenicol acetyltransferase (CAT) transient expression vectors, pL-EPA and pL-LPA, that differ only in their polyadenylation signals were constructed by using the early and late polyadenylation signals, respectively. In transfections of Cos, CV-1P, or HeLa cells and subsequent Northern blot analysis of CAT-specific RNA, approximately five times more steady-state CAT mRNA was produced in transfections with pL-LPA than with pL-EPA. The basis for this difference was not related to the specific promoter used or to RNA stability. Overall, the difference in steady-state mRNA levels derived from the two plasmids appeared to be attributable to intrinsic properties of the two polyadenylation signals, resulting in distinctly different cleavage and polyadenylation efficiencies. Additionally, we found that the utilization of the late polyadenylation site was dramatically reduced by deletion of sequences between 48 and 29 nucleotides 5' of the AAUAAA hexanucleotide. This reduction of mRNA levels was shown not to be caused by altered stability of mutant precursor RNAs or mRNAs, suggesting that these upstream sequences constitute an element of the late polyadenylation signal and may cause, at least to some extent, the greater efficiency of utilization of the late polyadenylation site.

Activation of the transforming potential of the human fos proto-oncogene requires message stabilization and results in increased amounts of partially modified fos protein

1988 ◽  
Vol 8 (12) ◽  
pp. 5521-5527
Author(s):  
W M Lee ◽  
C Lin ◽  
T Curran
Keyword(s):  
Leukemia Virus ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Moloney Murine Leukemia Virus ◽  
Coding Region ◽  
Serum Stimulation ◽  
Fos Protein ◽  
Steady State Levels ◽  
Message Stabilization

The requirements for activation of the transformation potential of the human c-fos proto-oncogene were investigated. Recombinant plasmids containing the Moloney murine leukemia virus long terminal repeat directing transcription of the c-fos coding region and either the authentic c-fos 3' untranslated region (UTR) or the 3' UTR from human c-myc were inefficient at inducing transformation. In contrast, a recombinant that substituted most of the c-fos 3' UTR with the 3' portion of the simian virus 40 T-antigen gene transformed cells well. This difference in transformation efficiency appeared to be due to significantly higher levels of fos mRNA and protein expressed from the transforming recombinant. This, in turn, was due to the much greater stability of its mRNA compared with those from the poorly transforming recombinants containing the c-fos or c-myc 3' UTR. Thus, the 3' UTR of the human c-fos mRNA is responsible for its rapid degradation and limits the steady-state levels of transcript and protein. Cells transformed by the activated human c-fos plasmids contained increased amounts of partially modified c-fos protein (c-Fos). This form of c-Fos turned over much more rapidly than the highly modified form of c-Fos induced by serum stimulation.

scholarly journals Induction of Endothelin-2 Expression by Luteinizing Hormone and Hypoxia: Possible Role in Bovine Corpus Luteum Formation

Endocrinology ◽  
2010 ◽  
Vol 151 (4) ◽  
pp. 1914-1922 ◽  
Author(s):  
Eyal Klipper ◽  
Anat Levit ◽  
Yonit Mastich ◽  
Bajram Berisha ◽  
Dieter Schams ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Corpus Luteum ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Low Oxygen ◽  
Vegf Mrna

The pattern and regulation of endothlin-2 (EDN2) expression and its putative roles in bovine ovaries were investigated. EDN2 mRNA was determined in corpus luteum (CL) and during folliculoluteal transition induced by GnRH in vivo. EDN2 was elevated only in the early CL and was not present in older CL. In the young CL, EDN2 mRNA was identified mainly in luteal cells but not endothelial cells that expressed the EDN1 gene. Similarly, in preovulatory follicles, EDN2 was expressed in the granulosa cells (GCs) and not in the vascular theca interna. LH and hypoxia are two major stimulants of CL formation. Therefore, GCs were cultured with bovine LH, under hypoxic conditions. GCs incubated with bovine LH resulted in increased EDN2 mRNA 42 h later. CoCl2, a hypoxia-mimicking agent, elevated EDN2 in GCs in a dose-dependent manner. Incubation of the human GC line (Simian virus 40 large T antigen) under low oxygen tension (1%) augmented EDN2 6 and 24 h later. In these two cell types, along with EDN2, hypoxia augmented VEGF. EDN2 induced in GCs changes that characterize the developing CL: cell proliferation as well as up-regulation of vascular endothelial growth factor and cyclooxygenase-2 (mRNA and protein levels). Human chorionic gonadotropin also up-regulated these two genes. Small interfering RNA targeting EDN-converting enzyme-1 effectively reduced its mRNA levels. This treatment, expected to lower the mature EDN2 peptide production, inhibited VEGF mRNA levels and GC numbers. Together these data suggest that elevated EDN2 in the early bovine CL, triggered by LH surge and hypoxia, may facilitate CL formation by promoting angiogenesis, cell proliferation, and differentiation.

scholarly journals Activation of the transforming potential of the human fos proto-oncogene requires message stabilization and results in increased amounts of partially modified fos protein.

1988 ◽  
Vol 8 (12) ◽  
pp. 5521-5527 ◽  
Author(s):  
W M Lee ◽  
C Lin ◽  
T Curran
Keyword(s):  
Leukemia Virus ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Moloney Murine Leukemia Virus ◽  
Coding Region ◽  
Serum Stimulation ◽  
Fos Protein ◽  
Steady State Levels ◽  
Message Stabilization

The requirements for activation of the transformation potential of the human c-fos proto-oncogene were investigated. Recombinant plasmids containing the Moloney murine leukemia virus long terminal repeat directing transcription of the c-fos coding region and either the authentic c-fos 3' untranslated region (UTR) or the 3' UTR from human c-myc were inefficient at inducing transformation. In contrast, a recombinant that substituted most of the c-fos 3' UTR with the 3' portion of the simian virus 40 T-antigen gene transformed cells well. This difference in transformation efficiency appeared to be due to significantly higher levels of fos mRNA and protein expressed from the transforming recombinant. This, in turn, was due to the much greater stability of its mRNA compared with those from the poorly transforming recombinants containing the c-fos or c-myc 3' UTR. Thus, the 3' UTR of the human c-fos mRNA is responsible for its rapid degradation and limits the steady-state levels of transcript and protein. Cells transformed by the activated human c-fos plasmids contained increased amounts of partially modified c-fos protein (c-Fos). This form of c-Fos turned over much more rapidly than the highly modified form of c-Fos induced by serum stimulation.

scholarly journals Activation of RNA Polymerase III Transcription in Cells Transformed by Simian Virus 40

1999 ◽  
Vol 19 (7) ◽  
pp. 4927-4934 ◽  
Author(s):  
Christopher G. C. Larminie ◽  
Josephine E. Sutcliffe ◽  
Kerrie Tosh ◽  
Andrew G. Winter ◽  
Zoe A. Felton-Edkins ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Mrna Levels ◽  
Large T Antigen ◽  
Tumor Viruses ◽  
Sv40 Transformation ◽  
Pol Iii

ABSTRACT RNA polymerase (Pol) III transcription is abnormally active in fibroblasts that have been transformed by simian virus 40 (SV40). This report presents evidence that two separate components of the general Pol III transcription apparatus, TFIIIB and TFIIIC2, are deregulated following SV40 transformation. TFIIIC2 subunits are expressed at abnormally high levels in SV40-transformed cells, an effect which is observed at both protein and mRNA levels. In untransformed fibroblasts, TFIIIB is subject to repression through association with the retinoblastoma protein RB. The interaction between RB and TFIIIB is compromised following SV40 transformation. Furthermore, the large T antigen of SV40 is shown to relieve repression by RB. The E7 oncoprotein of human papillomavirus can also activate Pol III transcription, an effect that is dependent on its ability to bind to RB. The data provide evidence that both TFIIIB and TFIIIC2 are targets for activation by DNA tumor viruses.

Decreased expression of protooncogenes c-fos, c-myc, and c-jun following polyamine depletion in IEC-6 cells

1993 ◽  
Vol 265 (2) ◽  
pp. G331-G338 ◽  
Author(s):  
J. Y. Wang ◽  
S. A. McCormack ◽  
M. J. Viar ◽  
H. Wang ◽  
C. Y. Tzen ◽  
...  
Keyword(s):  
Steady State ◽  
Specific Inhibitor ◽  
State Level ◽  
Cell Number ◽  
Mrna Levels ◽  
Direct Exposure ◽  
Mucosal Cells ◽  
Steady State Levels ◽  
Small Intestinal ◽  
Mucosal Growth

Direct exposure of small intestinal mucosal cells to luminal polyamines stimulates proliferation. This study tests the hypothesis that the protooncogenes c-fos, c-myc, c-jun, and junB are involved in the mechanism by which polyamines modulate mucosal growth. Studies were conducted in the IEC-6 cell line, derived from rat small intestinal crypt cells. Cells were grown in Dulbecco's minimal essential medium containing 5% dialyzed fetal bovine serum (dFBS) in the presence of absence of alpha-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase, which is the rate-limiting enzyme for polyamine synthesis. Cellular polyamine levels, cell growth, and relative abundance of c-fos, c-myc, c-jun, and junB mRNAs, were measured at 1, 2, 4, 6, 8, and 12 days after initial plating. The intracellular polyamines, spermidine and spermine, and their precursor, putrescine, in DFMO-treated cells decreased significantly at 2 days and remained depleted thereafter. Although DFMO profoundly decreased growth and final cell number, both control and DFMO-treated cells entered a plateau phase by 6 days. In control cells, c-myc and c-jun mRNA levels significantly increased on days 4-6 and then returned to a basal level of expression, which was maintained thereafter. c-fos mRNA in quiescent cells after 24 h serum deprivation was significantly stimulated by 5% dFBS, although a steady-state level of c-fos mRNA was undetectable in control cells. Treatment with DFMO not only prevented increased expression of c-myc and c-jun protooncogenes at 4 days, but also significantly reduced steady-state levels of c-myc and c-jun mRNA between 6 and 12 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of cellular thymidine kinase occurs at the mRNA level

1985 ◽  
Vol 5 (6) ◽  
pp. 1490-1497
Author(s):  
P Stuart ◽  
M Ito ◽  
C Stewart ◽  
S E Conrad
Keyword(s):  
Steady State ◽  
Thymidine Kinase ◽  
Cdna Clone ◽  
Blot Hybridization ◽  
Mrna Level ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Chromosomal Dna ◽  
Infected Cells ◽  
Steady State Levels

The thymidine kinase (TK) gene has been isolated from human genomic DNA. The gene was passaged twice by transfection of LTK- cells with human chromosomal DNA, and genomic libraries were made in lambda Charon 30 from a second-round TK+ transformant. When the library was screened with a human Alu probe, seven overlapping lambda clones from the human TK locus were obtained. None of the seven contained a functional TK gene as judged by transfection analysis, but several combinations of clones gave rise to TK+ colonies when cotransfected into TK- cells. A functional cDNA clone encoding the human TK gene has also been isolated. Using this cDNA clone as a probe in restriction enzyme/blot hybridization analyses, we have mapped the coding sequences and direction of transcription of the gene. We have also used a single-copy subclone from within the coding region to monitor steady-state levels of TK mRNA in serum-stimulated and simian virus 40-infected simian CV1 tissue culture cells. Our results indicate that the previously reported increase in TK enzyme levels seen after either treatment is paralleled by an equivalent increase in the steady-state levels of TK mRNA. In the case of simian virus 40-infected cells, the induction was delayed by 8 to 12 h, which is the length of time after infection required for early viral protein synthesis. In both cases, induction of TK mRNA coincides with the onset of DNA synthesis, but virally infected cells ultimately accumulate more TK mRNA than do serum-stimulated cells.

scholarly journals Hormonal regulation of collagenolysis in uterine cervical fibroblasts. Modulation of synthesis of procollagenase, prostromelysin and tissue inhibitor of metalloproteinases (TIMP) by progesterone and oestradiol-17 β

1991 ◽  
Vol 275 (3) ◽  
pp. 645-650 ◽  
Author(s):  
T Sato ◽  
A Ito ◽  
Y Mori ◽  
K Yamashita ◽  
T Hayakawa ◽  
...  
Keyword(s):  
Steady State ◽  
Hormonal Regulation ◽  
Culture Media ◽  
Interleukin 1 ◽  
State Level ◽  
The Other ◽  
Steady State Level ◽  
Tissue Inhibitor Of Metalloproteinases ◽  
Tissue Inhibitor ◽  
Steady State Levels

Rabbit uterine cervical fibroblasts produced a large amount of matrix metalloproteinases (MMPs) such as collagenase (MMP-1) and stromelysin (MMP-3) and a small relatively amount of tissue inhibitor of metalloproteinases (TIMP). When cells were treated with progesterone or oestradiol-17 beta, both steroids concurrently decreased the level of procollagenase and prostromelysin in the culture media and the steady-state levels of the respective mRNAs. On the other hand, the level of TIMP in the culture media and the steady-state level of its mRNA were simultaneously increased by these steroids. Similarly, the suppression of production of MMPs and the augmentation of TIMP production by both steroids were observed with interleukin 1 (IL-1)-treated cells, but the action of progesterone was more effective than that of oestradiol-17 beta in the IL-1-untreated and -treated cells. These results suggest that collagenolysis in uterine cervical fibroblasts is negatively regulated by steroid hormones via the acceleration of TIMP production and the suppression of synthesis of MMPs at the pretranslational level.

Efficiency of utilization of the simian virus 40 late polyadenylation site: effects of upstream sequences

1989 ◽  
Vol 9 (10) ◽  
pp. 4248-4258
Author(s):  
S Carswell ◽  
J C Alwine
Keyword(s):  
Steady State ◽  
Rna Stability ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Polyadenylation Signal ◽  
Polyadenylation Site ◽  
Expression Vectors ◽  
Mrna Levels ◽  
The Difference ◽  
Polyadenylation Signals

The late polyadenylation signal of simian virus 40 functions with greater efficiency than the early polyadenylation signal, in turn affecting steady-state mRNA levels. Two chloramphenicol acetyltransferase (CAT) transient expression vectors, pL-EPA and pL-LPA, that differ only in their polyadenylation signals were constructed by using the early and late polyadenylation signals, respectively. In transfections of Cos, CV-1P, or HeLa cells and subsequent Northern blot analysis of CAT-specific RNA, approximately five times more steady-state CAT mRNA was produced in transfections with pL-LPA than with pL-EPA. The basis for this difference was not related to the specific promoter used or to RNA stability. Overall, the difference in steady-state mRNA levels derived from the two plasmids appeared to be attributable to intrinsic properties of the two polyadenylation signals, resulting in distinctly different cleavage and polyadenylation efficiencies. Additionally, we found that the utilization of the late polyadenylation site was dramatically reduced by deletion of sequences between 48 and 29 nucleotides 5' of the AAUAAA hexanucleotide. This reduction of mRNA levels was shown not to be caused by altered stability of mutant precursor RNAs or mRNAs, suggesting that these upstream sequences constitute an element of the late polyadenylation signal and may cause, at least to some extent, the greater efficiency of utilization of the late polyadenylation site.

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