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Human T-lymphotropic virus type-1 p30 alters cell cycle G2 regulation of T lymphocytes to enhance cell survival.

Datta A, Silverman L, Phipps AJ, Hiraragi H, Ratner L, Lairmore MD - Retrovirology (2007)

Bottom Line: We then analyzed key proteins involved in G2-M checkpoint activation. p30 expression in Jurkat T-cells resulted in an increase in phosphorylation at serine 216 of nuclear cell division cycle 25C (Cdc25C), had enhanced checkpoint kinase 1 (Chk1) serine 345 phosphorylation, reduced expression of polo-like kinase 1 (PLK1), diminished phosphorylation of PLK1 at tyrosine 210 and reduced phosphorylation of Cdc25C at serine 198.Collectively these data are consistent with a cell survival role of p30 against genotoxic insults to HTLV-1 infected lymphocytes.Collectively, our data are the first to indicate that HTLV-1 p30 expression results in activation of the G2-M cell cycle checkpoint, events that would promote early viral spread and T-cell survival.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Retrovirus Research, The Ohio State University, Columbus, Ohio, USA. datta.15@osu.edu

ABSTRACT

Background: Human T-lymphotropic virus type-1 (HTLV-1) causes adult T-cell leukemia/lymphoma and is linked to a number of lymphocyte-mediated disorders. HTLV-1 contains both regulatory and accessory genes in four pX open reading frames. pX ORF-II encodes two proteins, p13 and p30, whose roles are still being defined in the virus life cycle and in HTLV-1 virus-host cell interactions. Proviral clones of HTLV-1 with pX ORF-II mutations diminish the ability of the virus to maintain viral loads in vivo. p30 expressed exogenously differentially modulates CREB and Tax-responsive element-mediated transcription through its interaction with CREB-binding protein/p300 and while acting as a repressor of many genes including Tax, in part by blocking tax/rex RNA nuclear export, selectively enhances key gene pathways involved in T-cell signaling/activation.

Results: Herein, we analyzed the role of p30 in cell cycle regulation. Jurkat T-cells transduced with a p30 expressing lentivirus vector accumulated in the G2-M phase of cell cycle. We then analyzed key proteins involved in G2-M checkpoint activation. p30 expression in Jurkat T-cells resulted in an increase in phosphorylation at serine 216 of nuclear cell division cycle 25C (Cdc25C), had enhanced checkpoint kinase 1 (Chk1) serine 345 phosphorylation, reduced expression of polo-like kinase 1 (PLK1), diminished phosphorylation of PLK1 at tyrosine 210 and reduced phosphorylation of Cdc25C at serine 198. Finally, primary human lymphocyte derived cell lines immortalized by a HTLV-1 proviral clone defective in p30 expression were more susceptible to camptothecin induced apoptosis. Collectively these data are consistent with a cell survival role of p30 against genotoxic insults to HTLV-1 infected lymphocytes.

Conclusion: Collectively, our data are the first to indicate that HTLV-1 p30 expression results in activation of the G2-M cell cycle checkpoint, events that would promote early viral spread and T-cell survival.

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HTLV-1 p30 does not modulate apoptosis in 293T cells or Jurkat T-cells. A) p30-expressing Jurkat T-cells or mock infected Jurkat T-cells were treated with camptothecin, etoposide, or TRAIL and assayed for apoptosis induction via Annexin V flow cytometry. Data represents the results of three independent experiments. Although camptothecin, etoposide, and TRAIL induced both cell lines into apoptosis, a differential degree of apoptosis induction was not seen between the two cell lines (nonparametric Wilcoxon rank sum test, p values: camptothecin 0.82, etoposide 0.51, TRAIL 0.13). Standard error bars are indicated. B) 293T cells were transiently transfected with either pME-p30HA or the empty pME-18S vector. Cells were untreated or treated with camptothecin or etoposide. Cell lysates were harvested and 50 μg of lysate was separated by SDS-PAGE. Apoptosis was assayed via immunoblot for the 89 kDa fragment of cleaved PARP. Expression of p30 was verified via immunoblot for HA. Expression of β-actin was verified as a loading control. - cells transfected with empty vector control; + cells transfected with pME-p30HA.
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Figure 4: HTLV-1 p30 does not modulate apoptosis in 293T cells or Jurkat T-cells. A) p30-expressing Jurkat T-cells or mock infected Jurkat T-cells were treated with camptothecin, etoposide, or TRAIL and assayed for apoptosis induction via Annexin V flow cytometry. Data represents the results of three independent experiments. Although camptothecin, etoposide, and TRAIL induced both cell lines into apoptosis, a differential degree of apoptosis induction was not seen between the two cell lines (nonparametric Wilcoxon rank sum test, p values: camptothecin 0.82, etoposide 0.51, TRAIL 0.13). Standard error bars are indicated. B) 293T cells were transiently transfected with either pME-p30HA or the empty pME-18S vector. Cells were untreated or treated with camptothecin or etoposide. Cell lysates were harvested and 50 μg of lysate was separated by SDS-PAGE. Apoptosis was assayed via immunoblot for the 89 kDa fragment of cleaved PARP. Expression of p30 was verified via immunoblot for HA. Expression of β-actin was verified as a loading control. - cells transfected with empty vector control; + cells transfected with pME-p30HA.

Mentions: We then tested the influence of exogenously expressed p30 on susceptibility of cells to apoptosis independent of other viral proteins. p30 was transiently expressed in Jurkat T-cells and 292T cells and tested for susceptibility to apoptotic stimuli. Expression of p30 in Jurkat T-cells did not result in increased apoptosis when left untreated, compared to mock infected cells, consistent with recent findings that p30 does not induce apoptosis in transiently transfected Molt-4 lymphocytes[15]. p30-expressing Jurkat T-cells and mock infected Jurkat T-cells were treated with camptothecin, etoposide, or TRAIL and assayed for apoptosis (Fig. 4A). Although the transduced cells were induced into apoptosis following treatment with camptothecin, etoposide, and TRAIL, there was no significant difference in the percentage of apoptotic cells between p30-expressing T-cells and mock Jurkat T-cells for any of the treatment groups (nonparametric Wilcoxon rank sum test, p values: camptothecin 0.82, etoposide 0.51, TRAIL 0.13). To examine the role of p30 in modulating cellular apoptosis in other cell types, we transiently transfected 293T cells with either pME-p30 HA or empty vector control (pME-18S). Following treatment with camptothecin or etoposide, cells were tested for apoptosis using immunoblot assay for the 89 kd fragment of cleaved PARP. Consistent with our data using Jurkat T-cells, we did not observe an increase in susceptibility to apoptosis between p30-expressing cells and negative control cells (Fig. 4B), and lead us to further test the influence of the viral protein in cell cycle regulation.


Human T-lymphotropic virus type-1 p30 alters cell cycle G2 regulation of T lymphocytes to enhance cell survival.

Datta A, Silverman L, Phipps AJ, Hiraragi H, Ratner L, Lairmore MD - Retrovirology (2007)

HTLV-1 p30 does not modulate apoptosis in 293T cells or Jurkat T-cells. A) p30-expressing Jurkat T-cells or mock infected Jurkat T-cells were treated with camptothecin, etoposide, or TRAIL and assayed for apoptosis induction via Annexin V flow cytometry. Data represents the results of three independent experiments. Although camptothecin, etoposide, and TRAIL induced both cell lines into apoptosis, a differential degree of apoptosis induction was not seen between the two cell lines (nonparametric Wilcoxon rank sum test, p values: camptothecin 0.82, etoposide 0.51, TRAIL 0.13). Standard error bars are indicated. B) 293T cells were transiently transfected with either pME-p30HA or the empty pME-18S vector. Cells were untreated or treated with camptothecin or etoposide. Cell lysates were harvested and 50 μg of lysate was separated by SDS-PAGE. Apoptosis was assayed via immunoblot for the 89 kDa fragment of cleaved PARP. Expression of p30 was verified via immunoblot for HA. Expression of β-actin was verified as a loading control. - cells transfected with empty vector control; + cells transfected with pME-p30HA.
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Figure 4: HTLV-1 p30 does not modulate apoptosis in 293T cells or Jurkat T-cells. A) p30-expressing Jurkat T-cells or mock infected Jurkat T-cells were treated with camptothecin, etoposide, or TRAIL and assayed for apoptosis induction via Annexin V flow cytometry. Data represents the results of three independent experiments. Although camptothecin, etoposide, and TRAIL induced both cell lines into apoptosis, a differential degree of apoptosis induction was not seen between the two cell lines (nonparametric Wilcoxon rank sum test, p values: camptothecin 0.82, etoposide 0.51, TRAIL 0.13). Standard error bars are indicated. B) 293T cells were transiently transfected with either pME-p30HA or the empty pME-18S vector. Cells were untreated or treated with camptothecin or etoposide. Cell lysates were harvested and 50 μg of lysate was separated by SDS-PAGE. Apoptosis was assayed via immunoblot for the 89 kDa fragment of cleaved PARP. Expression of p30 was verified via immunoblot for HA. Expression of β-actin was verified as a loading control. - cells transfected with empty vector control; + cells transfected with pME-p30HA.
Mentions: We then tested the influence of exogenously expressed p30 on susceptibility of cells to apoptosis independent of other viral proteins. p30 was transiently expressed in Jurkat T-cells and 292T cells and tested for susceptibility to apoptotic stimuli. Expression of p30 in Jurkat T-cells did not result in increased apoptosis when left untreated, compared to mock infected cells, consistent with recent findings that p30 does not induce apoptosis in transiently transfected Molt-4 lymphocytes[15]. p30-expressing Jurkat T-cells and mock infected Jurkat T-cells were treated with camptothecin, etoposide, or TRAIL and assayed for apoptosis (Fig. 4A). Although the transduced cells were induced into apoptosis following treatment with camptothecin, etoposide, and TRAIL, there was no significant difference in the percentage of apoptotic cells between p30-expressing T-cells and mock Jurkat T-cells for any of the treatment groups (nonparametric Wilcoxon rank sum test, p values: camptothecin 0.82, etoposide 0.51, TRAIL 0.13). To examine the role of p30 in modulating cellular apoptosis in other cell types, we transiently transfected 293T cells with either pME-p30 HA or empty vector control (pME-18S). Following treatment with camptothecin or etoposide, cells were tested for apoptosis using immunoblot assay for the 89 kd fragment of cleaved PARP. Consistent with our data using Jurkat T-cells, we did not observe an increase in susceptibility to apoptosis between p30-expressing cells and negative control cells (Fig. 4B), and lead us to further test the influence of the viral protein in cell cycle regulation.

Bottom Line: We then analyzed key proteins involved in G2-M checkpoint activation. p30 expression in Jurkat T-cells resulted in an increase in phosphorylation at serine 216 of nuclear cell division cycle 25C (Cdc25C), had enhanced checkpoint kinase 1 (Chk1) serine 345 phosphorylation, reduced expression of polo-like kinase 1 (PLK1), diminished phosphorylation of PLK1 at tyrosine 210 and reduced phosphorylation of Cdc25C at serine 198.Collectively these data are consistent with a cell survival role of p30 against genotoxic insults to HTLV-1 infected lymphocytes.Collectively, our data are the first to indicate that HTLV-1 p30 expression results in activation of the G2-M cell cycle checkpoint, events that would promote early viral spread and T-cell survival.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Retrovirus Research, The Ohio State University, Columbus, Ohio, USA. datta.15@osu.edu

ABSTRACT

Background: Human T-lymphotropic virus type-1 (HTLV-1) causes adult T-cell leukemia/lymphoma and is linked to a number of lymphocyte-mediated disorders. HTLV-1 contains both regulatory and accessory genes in four pX open reading frames. pX ORF-II encodes two proteins, p13 and p30, whose roles are still being defined in the virus life cycle and in HTLV-1 virus-host cell interactions. Proviral clones of HTLV-1 with pX ORF-II mutations diminish the ability of the virus to maintain viral loads in vivo. p30 expressed exogenously differentially modulates CREB and Tax-responsive element-mediated transcription through its interaction with CREB-binding protein/p300 and while acting as a repressor of many genes including Tax, in part by blocking tax/rex RNA nuclear export, selectively enhances key gene pathways involved in T-cell signaling/activation.

Results: Herein, we analyzed the role of p30 in cell cycle regulation. Jurkat T-cells transduced with a p30 expressing lentivirus vector accumulated in the G2-M phase of cell cycle. We then analyzed key proteins involved in G2-M checkpoint activation. p30 expression in Jurkat T-cells resulted in an increase in phosphorylation at serine 216 of nuclear cell division cycle 25C (Cdc25C), had enhanced checkpoint kinase 1 (Chk1) serine 345 phosphorylation, reduced expression of polo-like kinase 1 (PLK1), diminished phosphorylation of PLK1 at tyrosine 210 and reduced phosphorylation of Cdc25C at serine 198. Finally, primary human lymphocyte derived cell lines immortalized by a HTLV-1 proviral clone defective in p30 expression were more susceptible to camptothecin induced apoptosis. Collectively these data are consistent with a cell survival role of p30 against genotoxic insults to HTLV-1 infected lymphocytes.

Conclusion: Collectively, our data are the first to indicate that HTLV-1 p30 expression results in activation of the G2-M cell cycle checkpoint, events that would promote early viral spread and T-cell survival.

Show MeSH
Related in: MedlinePlus