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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

Low Dlg1 expression in IL-2-independent Tax1ΔC clones. CTLL-2 cells were infected with Tax1-virus (lanes 1–5) or Tax1ΔC-virus (lanes 6–10), and seeded in 96-well plates in the absence IL-2 for more than one month. The expression of Dlg1 (top), Syntrophin β (second column), Tax1 (third column), or Tubulin (bottom) in Tax1-transformed CTLL-2 clones (lanes 5–8) and Tax1ΔC-transformed clones (lane 9–12), was measured by Western blot analysis using corresponding antibodies.
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Figure 5: Low Dlg1 expression in IL-2-independent Tax1ΔC clones. CTLL-2 cells were infected with Tax1-virus (lanes 1–5) or Tax1ΔC-virus (lanes 6–10), and seeded in 96-well plates in the absence IL-2 for more than one month. The expression of Dlg1 (top), Syntrophin β (second column), Tax1 (third column), or Tubulin (bottom) in Tax1-transformed CTLL-2 clones (lanes 5–8) and Tax1ΔC-transformed clones (lane 9–12), was measured by Western blot analysis using corresponding antibodies.

Mentions: To confirm the effect of D1g1 knockdown, we examined the expression of Dlg1 in the cells characterized above. Western blot analysis with anti-Dlg1 antibody demonstrated reduced expression of Dlg1 in the Dlg1 knockdown cells (Dlg1-1, Dlg1-3) even after IL-2-independent transformation by Tax1 or Tax1ΔC (Figure 4A). Interestingly, IL-2-independent control cells (CAT, LUC) transformed either by Tax1ΔC or Tax1 expressed less Dlg1 compared to the parental non-transformed cells, and the reduction of Dlg1 was much more prominent in Tax1ΔC cells relative to Tax1. These results support a hypothesis that IL-2-independent transformation of CTLL-2 cells by Tax1ΔC requires reduction of Dlg1 expression. In the above experiments, we used bulk (non-clonal) CTLL-2 cells transformed by Tax1ΔC or Tax1, which were established in culture flasks. To examine this hypothesis further, we evaluated five independent clonal CTLL-2 cell lines transformed either by Tax1ΔC or Tax1 established in 96-well plates and compared the expression level of Dlg1 protein in these cloned cells (Fig 5). All five IL-2-independent Tax1ΔC clones expressed reduced amounts of Dlg1, whereas two out of five Tax1 clones expressed Dlg1 at a level similar to Tax1ΔC. The expression of Syntrophin β, another PDZ protein suggested to interact with Tax1 [27], was expressed equivalently in these cell lines, indicating that reduced expression of Dlg1 in Tax1ΔC is specific. These data lend additional support to the hypothesis that reduced expression of Dlg1 protein is a factor required for Tax1ΔC-induced IL-2-independent transformation of CTLL-2 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)

Low Dlg1 expression in IL-2-independent Tax1ΔC clones. CTLL-2 cells were infected with Tax1-virus (lanes 1–5) or Tax1ΔC-virus (lanes 6–10), and seeded in 96-well plates in the absence IL-2 for more than one month. The expression of Dlg1 (top), Syntrophin β (second column), Tax1 (third column), or Tubulin (bottom) in Tax1-transformed CTLL-2 clones (lanes 5–8) and Tax1ΔC-transformed clones (lane 9–12), was measured by Western blot analysis using corresponding antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Low Dlg1 expression in IL-2-independent Tax1ΔC clones. CTLL-2 cells were infected with Tax1-virus (lanes 1–5) or Tax1ΔC-virus (lanes 6–10), and seeded in 96-well plates in the absence IL-2 for more than one month. The expression of Dlg1 (top), Syntrophin β (second column), Tax1 (third column), or Tubulin (bottom) in Tax1-transformed CTLL-2 clones (lanes 5–8) and Tax1ΔC-transformed clones (lane 9–12), was measured by Western blot analysis using corresponding antibodies.
Mentions: To confirm the effect of D1g1 knockdown, we examined the expression of Dlg1 in the cells characterized above. Western blot analysis with anti-Dlg1 antibody demonstrated reduced expression of Dlg1 in the Dlg1 knockdown cells (Dlg1-1, Dlg1-3) even after IL-2-independent transformation by Tax1 or Tax1ΔC (Figure 4A). Interestingly, IL-2-independent control cells (CAT, LUC) transformed either by Tax1ΔC or Tax1 expressed less Dlg1 compared to the parental non-transformed cells, and the reduction of Dlg1 was much more prominent in Tax1ΔC cells relative to Tax1. These results support a hypothesis that IL-2-independent transformation of CTLL-2 cells by Tax1ΔC requires reduction of Dlg1 expression. In the above experiments, we used bulk (non-clonal) CTLL-2 cells transformed by Tax1ΔC or Tax1, which were established in culture flasks. To examine this hypothesis further, we evaluated five independent clonal CTLL-2 cell lines transformed either by Tax1ΔC or Tax1 established in 96-well plates and compared the expression level of Dlg1 protein in these cloned cells (Fig 5). All five IL-2-independent Tax1ΔC clones expressed reduced amounts of Dlg1, whereas two out of five Tax1 clones expressed Dlg1 at a level similar to Tax1ΔC. The expression of Syntrophin β, another PDZ protein suggested to interact with Tax1 [27], was expressed equivalently in these cell lines, indicating that reduced expression of Dlg1 in Tax1ΔC is specific. These data lend additional support to the hypothesis that reduced expression of Dlg1 protein is a factor required for Tax1ΔC-induced IL-2-independent transformation of CTLL-2 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