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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.

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Related in: MedlinePlus

Dlg1 knockdown augments IL-2-independent cell growth induced by Tax1. (A) CTLL-2 cells were infected with a lentivirus encoding Tax1. Forty-eight hours after infection, cell lysates were prepared and the amount of Tax1 in the lysates was measured by Western blot analysis using an anti-Tax1 antibody. (B) CTLL-2 cells (Dlg1-1, Dlg1-3, CAT, LUC) infected with Tax1-virus were washed twice with PBS, seeded into 96-well plates at 3 × 102 cells per well, and cultured in the absence of IL-2 for four weeks. The number of wells containing outgrowing cells was counted under a light microscope. Transformation efficiency (%) was calculated as a ratio of the number of positive wells out of 96 wells. Error bars indicate standard deviations in three independent experiments.
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Figure 2: Dlg1 knockdown augments IL-2-independent cell growth induced by Tax1. (A) CTLL-2 cells were infected with a lentivirus encoding Tax1. Forty-eight hours after infection, cell lysates were prepared and the amount of Tax1 in the lysates was measured by Western blot analysis using an anti-Tax1 antibody. (B) CTLL-2 cells (Dlg1-1, Dlg1-3, CAT, LUC) infected with Tax1-virus were washed twice with PBS, seeded into 96-well plates at 3 × 102 cells per well, and cultured in the absence of IL-2 for four weeks. The number of wells containing outgrowing cells was counted under a light microscope. Transformation efficiency (%) was calculated as a ratio of the number of positive wells out of 96 wells. Error bars indicate standard deviations in three independent experiments.

Mentions: To examine the roles of Dlg1 protein in Tax1-induced IL-2-independent growth of CTLL-2 cells, we established CTLL-2 cells expressing reduced amount of Dlg1 using RNA interference (RNAi). We first constructed lentivirus vectors expressing short hairpin (sh)RNA specific to mouse dlg1 sequences (Dlg1-1, Dlg1-3). Dlg1-1 and Dlg1-3 target distinct sequences of mouse Dlg1 RNA. Two control shRNAs were constructed to target bacterial chloramphenicol acetyltransferase (CAT) and renilla luciferase (LUC) genes, both of which are not expressed normally in mouse T-cells. These viruses were used to infect CTLL-2 cells, and the infected cells were selected by blasticidin for more than 10 days. The established cell lines then were examined for the expression of Dlg1 protein by Western blotting analysis with anti-Dlg1 antibody (Figure 1). Two Dlg1 knockdown cell lines (Dlg1-1, Dlg1-3) expressed a reduced amount of Dlg1 protein relative to two control cell lines (CAT, LUC). These four cell lines grew at equivalent rates in the presence of IL-2 (Figure 1B), and died without IL-2 with similar kinetics (data not shown). Thus, the reduction of Dlg1 expression in CTLL-2 cells did not affect apparent cell growth phenotypes. To examine the effect of Dlg1 knockdown on Tax1-induced IL-2-independent growth, these characterized Dlg1 knockdown cell lines were infected with a lentivirus expressing Tax1 (Tax1-virus) and cultured in the presence of IL-2 for 48 h. Subsequently, the cells were seeded into 96-well plates, followed by further culturing in the absence of IL-2. After more than two weeks, the number of wells with outgrowing CTLL-2 cells was counted using a microscope. All CTLL-2 cells infected with a control lentivirus died within two weeks and did not induce any outgrowing cells (data not shown). Conversely, there was an outgrowth of control CTLL-2 infected with the Tax1-virus (CAT/Tax1, LUC/Tax1) at 7–11% of wells (Figure 2). Similarly, Dlg1 knockdown cells infected with the Tax1-virus (Dlg1-1, Dlg1-3) also had outgrowth with three to six-folds more wells than the controls (CAT/Tax1, LUC/Tax1). The observed differences were not due to reduced Tax1 expression in the control cells, since all four cell lines expressed equivalent amounts of Tax1 protein after the infection as shown by Western analysis (Figure 2A). The augmented Tax1 activity was reproducibly observed in at least nine independent experiments (data not shown). The Dlg1-1 and Dlg1-3 cell lines established by independent experiments also reproduced the high sensitivity to Tax1 transformation relative to control cells (data not shown). Taken together, these results indicate that the reduction of Dlg1 protein in CTLL-2 augmented the ability of Tax1 to induce IL-2 independent growth.


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 augments IL-2-independent cell growth induced by Tax1. (A) CTLL-2 cells were infected with a lentivirus encoding Tax1. Forty-eight hours after infection, cell lysates were prepared and the amount of Tax1 in the lysates was measured by Western blot analysis using an anti-Tax1 antibody. (B) CTLL-2 cells (Dlg1-1, Dlg1-3, CAT, LUC) infected with Tax1-virus were washed twice with PBS, seeded into 96-well plates at 3 × 102 cells per well, and cultured in the absence of IL-2 for four weeks. The number of wells containing outgrowing cells was counted under a light microscope. Transformation efficiency (%) was calculated as a ratio of the number of positive wells out of 96 wells. Error bars indicate standard deviations in three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Dlg1 knockdown augments IL-2-independent cell growth induced by Tax1. (A) CTLL-2 cells were infected with a lentivirus encoding Tax1. Forty-eight hours after infection, cell lysates were prepared and the amount of Tax1 in the lysates was measured by Western blot analysis using an anti-Tax1 antibody. (B) CTLL-2 cells (Dlg1-1, Dlg1-3, CAT, LUC) infected with Tax1-virus were washed twice with PBS, seeded into 96-well plates at 3 × 102 cells per well, and cultured in the absence of IL-2 for four weeks. The number of wells containing outgrowing cells was counted under a light microscope. Transformation efficiency (%) was calculated as a ratio of the number of positive wells out of 96 wells. Error bars indicate standard deviations in three independent experiments.
Mentions: To examine the roles of Dlg1 protein in Tax1-induced IL-2-independent growth of CTLL-2 cells, we established CTLL-2 cells expressing reduced amount of Dlg1 using RNA interference (RNAi). We first constructed lentivirus vectors expressing short hairpin (sh)RNA specific to mouse dlg1 sequences (Dlg1-1, Dlg1-3). Dlg1-1 and Dlg1-3 target distinct sequences of mouse Dlg1 RNA. Two control shRNAs were constructed to target bacterial chloramphenicol acetyltransferase (CAT) and renilla luciferase (LUC) genes, both of which are not expressed normally in mouse T-cells. These viruses were used to infect CTLL-2 cells, and the infected cells were selected by blasticidin for more than 10 days. The established cell lines then were examined for the expression of Dlg1 protein by Western blotting analysis with anti-Dlg1 antibody (Figure 1). Two Dlg1 knockdown cell lines (Dlg1-1, Dlg1-3) expressed a reduced amount of Dlg1 protein relative to two control cell lines (CAT, LUC). These four cell lines grew at equivalent rates in the presence of IL-2 (Figure 1B), and died without IL-2 with similar kinetics (data not shown). Thus, the reduction of Dlg1 expression in CTLL-2 cells did not affect apparent cell growth phenotypes. To examine the effect of Dlg1 knockdown on Tax1-induced IL-2-independent growth, these characterized Dlg1 knockdown cell lines were infected with a lentivirus expressing Tax1 (Tax1-virus) and cultured in the presence of IL-2 for 48 h. Subsequently, the cells were seeded into 96-well plates, followed by further culturing in the absence of IL-2. After more than two weeks, the number of wells with outgrowing CTLL-2 cells was counted using a microscope. All CTLL-2 cells infected with a control lentivirus died within two weeks and did not induce any outgrowing cells (data not shown). Conversely, there was an outgrowth of control CTLL-2 infected with the Tax1-virus (CAT/Tax1, LUC/Tax1) at 7–11% of wells (Figure 2). Similarly, Dlg1 knockdown cells infected with the Tax1-virus (Dlg1-1, Dlg1-3) also had outgrowth with three to six-folds more wells than the controls (CAT/Tax1, LUC/Tax1). The observed differences were not due to reduced Tax1 expression in the control cells, since all four cell lines expressed equivalent amounts of Tax1 protein after the infection as shown by Western analysis (Figure 2A). The augmented Tax1 activity was reproducibly observed in at least nine independent experiments (data not shown). The Dlg1-1 and Dlg1-3 cell lines established by independent experiments also reproduced the high sensitivity to Tax1 transformation relative to control cells (data not shown). Taken together, these results indicate that the reduction of Dlg1 protein in CTLL-2 augmented the ability of Tax1 to induce IL-2 independent growth.

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