Limits...
Inactivation of tumor suppressor Dlg1 augments transformation of a T-cell line induced by human T-cell leukemia virus type 1 Tax protein.

Ishioka K, Higuchi M, Takahashi M, Yoshida S, Oie M, Tanaka Y, Takahashi S, Xie L, Green PL, Fujii M - Retrovirology (2006)

Bottom Line: A Tax1 mutant defective for the Dlg1 interaction showed reduced transformation of CTLL-2 compared to wild type Tax1, but the transformation was minimally affected by Dlg1 reduction.Moreover, all human T-cell lines immortalized by HTLV-1, including the recombinant HTLV-1-containing Tax1DeltaC, expressed less Dlg1 than control T-cell lines.These results suggest that inactivation of Dlg1 augments Tax1-mediated transformation of CTLL-2, and PDZ protein(s) other than Dlg1 are critically involved in the transformation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Virology, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-Dori, Niigata, Japan. kojiro1@med.niigata-u.ac.jp

ABSTRACT

Background: The interaction of human T-cell leukemia virus type 1 (HTLV-1) Tax1 protein with the tumor suppressor Dlg1 is correlated with cellular transformation.

Results: Here, we show that Dlg1 knockdown by RNA interference increases the ability of Tax1 to transform a mouse T-cell line (CTLL-2), as measured interleukin (IL)-2-independent growth. A Tax1 mutant defective for the Dlg1 interaction showed reduced transformation of CTLL-2 compared to wild type Tax1, but the transformation was minimally affected by Dlg1 reduction. The few Tax1DeltaC-transduced CTLL-2 cells that became transformed expressed less Dlg1 than parental cells, suggesting that Dlg1-low cells were selectively transformed by Tax1DeltaC. Moreover, all human T-cell lines immortalized by HTLV-1, including the recombinant HTLV-1-containing Tax1DeltaC, expressed less Dlg1 than control T-cell lines.

Conclusion: These results suggest that inactivation of Dlg1 augments Tax1-mediated transformation of CTLL-2, and PDZ protein(s) other than Dlg1 are critically involved in the transformation.

Show MeSH

Related in: MedlinePlus

Dlg1 knockdown little affects transcriptional activity of Tax1. (A) Cell lysates were prepared from the indicated knockdown cells (hDlg1-1, hDlg1-3, Rluc), and the amounts of hDlg1 protein and Tubulin in cell lysates were measured by Western blot analysis using anti-hDlg1 antibody (top) and anti-Tubulin (bottom), respectively. (B) Jurkat cells (hDlg1-1, hDlg1-3, Rluc) were transfected with κ B-Luc plasmid together with pHβPr-1-Tax1-neo plasmid using the lipofection method. Forty-eight hours after transfection, cell lysates were prepared and the luciferase activity in the lysates was measured by a luminometer. Error bars indicate standard deviations.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC1622753&req=5

Figure 7: Dlg1 knockdown little affects transcriptional activity of Tax1. (A) Cell lysates were prepared from the indicated knockdown cells (hDlg1-1, hDlg1-3, Rluc), and the amounts of hDlg1 protein and Tubulin in cell lysates were measured by Western blot analysis using anti-hDlg1 antibody (top) and anti-Tubulin (bottom), respectively. (B) Jurkat cells (hDlg1-1, hDlg1-3, Rluc) were transfected with κ B-Luc plasmid together with pHβPr-1-Tax1-neo plasmid using the lipofection method. Forty-eight hours after transfection, cell lysates were prepared and the luciferase activity in the lysates was measured by a luminometer. Error bars indicate standard deviations.

Mentions: We next examined the effect of Dlg1 knockdown on Tax1 transcriptional activity. To do so, Jurkat cells were infected with lentivirus expressing shRNA against human dlg1 (hDlg1). Western blotting analysis showed that two hDlg1 knockdown cell lines (hDlg1-1, hDlg1-3) expressed a reduced amount of Dlg1 protein relative to a control cell line (Rluc) targeting a renilla luciferase gene (Figure 7A). These cell lines were then transfected with a Tax1 expression plasmid together with a firefly luciferase reporter plasmid regulated by the NF-κB site, which acts as a Tax1-inducible element, by the lipofection method. Tax1 efficiently activated NF-κB -dependent luciferase activity in two hDlg1 knockdown cells, and the activities were equivalent to those in the control cells (Rluc, None). These results indicates that reduction of hDlg1 protein little affects Tax1 dependent NF-κB activation in T-cells.


Inactivation of tumor suppressor Dlg1 augments transformation of a T-cell line induced by human T-cell leukemia virus type 1 Tax protein.

Ishioka K, Higuchi M, Takahashi M, Yoshida S, Oie M, Tanaka Y, Takahashi S, Xie L, Green PL, Fujii M - Retrovirology (2006)

Dlg1 knockdown little affects transcriptional activity of Tax1. (A) Cell lysates were prepared from the indicated knockdown cells (hDlg1-1, hDlg1-3, Rluc), and the amounts of hDlg1 protein and Tubulin in cell lysates were measured by Western blot analysis using anti-hDlg1 antibody (top) and anti-Tubulin (bottom), respectively. (B) Jurkat cells (hDlg1-1, hDlg1-3, Rluc) were transfected with κ B-Luc plasmid together with pHβPr-1-Tax1-neo plasmid using the lipofection method. Forty-eight hours after transfection, cell lysates were prepared and the luciferase activity in the lysates was measured by a luminometer. Error bars indicate standard deviations.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1622753&req=5

Figure 7: Dlg1 knockdown little affects transcriptional activity of Tax1. (A) Cell lysates were prepared from the indicated knockdown cells (hDlg1-1, hDlg1-3, Rluc), and the amounts of hDlg1 protein and Tubulin in cell lysates were measured by Western blot analysis using anti-hDlg1 antibody (top) and anti-Tubulin (bottom), respectively. (B) Jurkat cells (hDlg1-1, hDlg1-3, Rluc) were transfected with κ B-Luc plasmid together with pHβPr-1-Tax1-neo plasmid using the lipofection method. Forty-eight hours after transfection, cell lysates were prepared and the luciferase activity in the lysates was measured by a luminometer. Error bars indicate standard deviations.
Mentions: We next examined the effect of Dlg1 knockdown on Tax1 transcriptional activity. To do so, Jurkat cells were infected with lentivirus expressing shRNA against human dlg1 (hDlg1). Western blotting analysis showed that two hDlg1 knockdown cell lines (hDlg1-1, hDlg1-3) expressed a reduced amount of Dlg1 protein relative to a control cell line (Rluc) targeting a renilla luciferase gene (Figure 7A). These cell lines were then transfected with a Tax1 expression plasmid together with a firefly luciferase reporter plasmid regulated by the NF-κB site, which acts as a Tax1-inducible element, by the lipofection method. Tax1 efficiently activated NF-κB -dependent luciferase activity in two hDlg1 knockdown cells, and the activities were equivalent to those in the control cells (Rluc, None). These results indicates that reduction of hDlg1 protein little affects Tax1 dependent NF-κB activation in T-cells.

Bottom Line: A Tax1 mutant defective for the Dlg1 interaction showed reduced transformation of CTLL-2 compared to wild type Tax1, but the transformation was minimally affected by Dlg1 reduction.Moreover, all human T-cell lines immortalized by HTLV-1, including the recombinant HTLV-1-containing Tax1DeltaC, expressed less Dlg1 than control T-cell lines.These results suggest that inactivation of Dlg1 augments Tax1-mediated transformation of CTLL-2, and PDZ protein(s) other than Dlg1 are critically involved in the transformation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Virology, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-Dori, Niigata, Japan. kojiro1@med.niigata-u.ac.jp

ABSTRACT

Background: The interaction of human T-cell leukemia virus type 1 (HTLV-1) Tax1 protein with the tumor suppressor Dlg1 is correlated with cellular transformation.

Results: Here, we show that Dlg1 knockdown by RNA interference increases the ability of Tax1 to transform a mouse T-cell line (CTLL-2), as measured interleukin (IL)-2-independent growth. A Tax1 mutant defective for the Dlg1 interaction showed reduced transformation of CTLL-2 compared to wild type Tax1, but the transformation was minimally affected by Dlg1 reduction. The few Tax1DeltaC-transduced CTLL-2 cells that became transformed expressed less Dlg1 than parental cells, suggesting that Dlg1-low cells were selectively transformed by Tax1DeltaC. Moreover, all human T-cell lines immortalized by HTLV-1, including the recombinant HTLV-1-containing Tax1DeltaC, expressed less Dlg1 than control T-cell lines.

Conclusion: These results suggest that inactivation of Dlg1 augments Tax1-mediated transformation of CTLL-2, and PDZ protein(s) other than Dlg1 are critically involved in the transformation.

Show MeSH
Related in: MedlinePlus