Characterization of functional domains necessary for mutant p53 gain of function

Abstract

Tumor cells, including SW480 carcinoma cells that carry a mutant p53, are addicted to the mutant for their survival and resistance to growth suppression by chemotherapeutic agents. Here, we investigated whether various classes of p53 mutants share a common property and functional domains necessary for mutant p53 gain of function. To test this, we generated SW480 cell lines in which endogenous mutant R273H/P309S can be inducibly or stably knocked down, whereas a small interfering RNA-resistant mutant p53 along with a mutated functional domain can be inducibly or stably expressed. We found that both contact-site (R248W and R273H) and conformation (G245S and R249S) mutants are able to maintain the transformed phenotypes of SW480 cells conferred by endogenous mutant p53. We also found that activation domains 1-2 and the proline-rich domain are required for mutant p53 gain of function. Interestingly, we showed that the C-terminal basic domain, which is required for wild-type p53 activity, is an inhibitory domain for mutant p53. Furthermore, we showed that deletion of the basic domain enhances, whereas a mutation in activation domains 1-2 and deletion of the proline-rich domain abolish mutant p53 to regulate Gro1 and Id2, both of which are regulated by and mediate endogenous mutant p53 gain of function. These results indicate that both conformation and contact-site mutants share a property for cell transformation, and the domains critical for wild-type p53 tumor suppression are also required for mutant p53 tumor promotion. Thus, the inhibitory basic domain and the common property for p53 mutants can be explored for targeting tumors with mutant p53.

FIGURE 1.

Mutant p53 is required for maintaining the transformed phenotypes of SW480 cells. A, Western blots were prepared with extracts from SW480 cells uninduced (−) or induced (+) to knock down mutant p53 for 3 days and then probed with antibodies against p53 and actin, respectively. B, left, colony formation assay was performed with SW480 cells uninduced or induced to knock down endogenous mutant p53 along with mock treatment or treatment with camptothecin (CPT). Right, quantification of the number of colonies with a diameter of >1 mm. *, p < 0.05. Error bars indicate S.E. C, generation of SW480 cell lines in which siRNA against bacterial LacZ can be inducibly expressed. Western blots were prepared with extracts from SW480 cells, which were transiently transfected with HA-tagged LacZ for 1 day and then uninduced (−) or induced (+) to express siRNA against LacZ mRNA for 3 days. The blots were then probed with antibodies against HA tag, p53, and actin, respectively. D, left, colony formation assay was performed with SW480 cells uninduced or induced to express siRNA against LacZ along with mock treatment or treatment with CPT. Right, quantification of the number of colonies with a diameter of >1 mm. Error bars indicate S.E.

FIGURE 2.

The proliferation defect of SW480 cells induced by knockdown of endogenous mutant p53 can be rescued by stable expression of an siRNA-resistant mutant p53. A, C, E, and G, generation of SW480 cell lines in which endogenous mutant p53 can be inducibly knocked down, whereas an siRNA-resistant mutant G245S (A), R248W (C), R249S (E), or R273H (G) was stably expressed. Western blots were prepared with extracts from SW480 cells uninduced (−) or induced (+) to knock down endogenous mutant p53 for 3 days and then probed with antibodies against p53 and actin, respectively. B, D, F, and H, left, SW480 cells, in which siRNA-resistant mutant G245S (B), R248W (D), R249S (F), or R273H (H) was stably expressed, were uninduced or induced to knock down endogenous mutant p53 along with mock treatment or treatment with CPT and then cultured for 14 days. Right, quantification of the number of colonies with a diameter of >1 mm. Error bars indicate S.E.

FIGURE 3.

Inducible expression of an siRNA-resistant mutant p53 promotes the colony-forming ability of SW480 cells in which endogenous mutant p53 was stably knocked down. A, C, E, and G, generation of SW480 cell lines in which endogenous mutant p53 was stably knocked down, whereas an siRNA-resistant mutant G245S (A), R248W (C), R249S (E), or R273H (G) can be inducibly expressed. Western blots were prepared with extracts from SW480 cells uninduced (−) or induced (+) to express an exogenous mutant p53 for 1 day and then probed with antibodies against p53 and actin, respectively. B, D, F, and H, left, SW480 cells, in which endogenous mutant p53 was stably knocked down, were uninduced or induced to express mutant G245S (B), R248W (D), R249S (F), or R273H (H) along with mock treatment or treatment with CPT and then cultured for 14 days. Right, quantification of the number of colonies with a diameter of >1 mm. *, p < 0.05. Error bars indicate S.E.

FIGURE 4.

The proliferation defect of SW480 cells induced by knockdown of endogenous mutant p53 can be rescued by inducible expression of an siRNA-resistant mutant p53. A and C, generation of SW480 cell lines in which endogenous mutant p53 can be inducibly knocked down, whereas siRNA-resistant mutant G245S (A) or R248W (C) can be inducibly expressed. Western blots were prepared with extracts from SW480 cells uninduced (−) or induced (+) to knock down endogenous mutant p53 and simultaneously express an exogenous mutant p53 for 1 day and then probed with antibodies against p53 and actin, respectively. B and D, left, SW480 cells were uninduced or induced to knock down endogenous mutant p53 and simultaneously express mutant G245S (B) or R248W (D) along with mock treatment or treatment with CPT and then cultured for 14 days. Right, quantification of the number of colonies with a diameter of >1 mm. *, p < 0.05. Error bars indicate S.E.

FIGURE 5.

Mutant G245S or R248W with a double-point mutation (Gln-22/Ser-23) in activation domain 1 is deficient in rescuing the proliferation defect of SW480 cells induced by knockdown of endogenous mutant p53. A and C, generation of SW480 cell lines in which endogenous mutant p53 can be inducibly knocked down, whereas siRNA-resistant mutant G245S(Gln-22/Ser-23) (A) or R248W(Gln-22/Ser-23) (C) can be inducibly expressed. The experiment was performed as in Fig. 4A. B and D, left, SW480 cells were uninduced or induced to knock down endogenous mutant p53 and simultaneously express mutant G245S(Gln-22/Ser-23) (B) or R248W(Gln-22/Ser-23) (D) along with mock treatment or treatment with CPT and then cultured for 14 days. Right, quantification of the number of colonies with a diameter of >1 mm. *, p < 0.05. Error bars indicate S.E.

FIGURE 6.

Mutant G245S or R248W with a double-point mutation (Gln-53/Ser-54) in activation domain 2 is deficient in rescuing the proliferation defect of SW480 cells induced by knockdown of endogenous mutant p53. A and C, generation of SW480 cell lines in which endogenous mutant p53 can be inducibly knocked down, whereas siRNA-resistant mutant G245S(Gln-53/Ser-54) (A) or R248W(Gln-53/Ser-54) (C) can be inducibly expressed. The experiment was performed as in Fig. 4A. B and D, left, SW480 cells were uninduced or induced to knock down endogenous mutant p53 and simultaneously express mutant G245S(Gln-53/Ser-54) (B) or R248W(Gln-53/Ser-54) (D) along with mock treatment or treatment with CPT and then cultured for 14 days. Right, quantification of the number of colonies with a diameter of >1 mm. *, p < 0.05. Error bars indicate S.E.

FIGURE 7.

Mutant G245S or R248W without the proline-rich domain (Δ62–91) is deficient in rescuing the proliferation defect of SW480 cells induced by knockdown of endogenous mutant p53. A and C, generation of SW480 cell lines in which endogenous mutant p53 can be inducibly knocked down, whereas siRNA-resistant mutant G245S(ΔPRD) (A) or R248W(ΔPRD) (C) can be inducibly expressed. The experiment was performed as in Fig. 4A. B and D, left, SW480 cells were uninduced or induced to knock down endogenous mutant p53 and simultaneously express mutant G245S(ΔPRD) (B) or R248W(ΔPRD) (D) along with mock treatment or treatment with CPT and then cultured for 14 days. Right, quantification of the number of colonies with a diameter of >1 mm. *, p < 0.05. Error bars indicate S.E.

FIGURE 8.

Mutant G245S or R248W without the C-terminal basic domain (Δ364–393) enhances the colony-forming ability of SW480 cells upon knockdown of endogenous mutant p53. A and C, generation of SW480 cell lines in which endogenous mutant p53 can be inducibly knocked down, whereas siRNA-resistant mutant G245S(ΔBD) (A) or R248W(ΔBD) (C) can be inducibly expressed. The experiment was performed as in Fig. 4A. B and D, left, SW480 cells were uninduced or induced to knock down endogenous mutant p53 and simultaneously express mutant G245S(ΔBD) (B) or R248W(ΔBD) (D) along with mock treatment or treatment with CPT and then cultured for 14 days. Right, quantification of the number of colonies with a diameter of >1 mm. *, p < 0.05. Error bars indicate S.E. E and G, generation of SW480 cell lines in which endogenous mutant p53 was stably knocked down, whereas siRNA-resistant mutant G245S(ΔBD) (E) or R248W(ΔBD) (G) can be inducibly expressed. The experiment was performed as in Fig. 3A. F and H, left, SW480 cells, in which endogenous mutant p53 was stably knocked down, were uninduced or induced to express mutant G245S(ΔBD) (F) or R248W(ΔBD) (H) along with mock treatment or treatment with CPT and then cultured for 14 days. Right, quantification of the number of colonies with a diameter of >1 mm. *, p < 0.05. Error bars indicate S.E.

FIGURE 9.

AD1, AD2, and PRD are necessary for mutant p53 to regulate Gro1 and Id2, whereas BD is an inhibitory domain. A, semiquantitative RT-PCR was performed to measure the level of Gro1 and Id2 in SW480 cells uninduced or induced to express siRNA against LacZ or knock down endogenous mutant p53 for 3 days. The level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was measured as loading control. B–F, the experiment was performed as in A except that SW480 cells were uninduced or induced to knock down endogenous mutant p53 and simultaneously express siRNA-resistant mutant G245S or R248W (B), G245S(Gln-22/Ser-23) or R248W(Gln-22/Ser-23) (C), G245S(Gln-53/Ser-54) or R248W(Gln-53/Ser-54) (D), G245S(ΔPRD) or R248W(ΔPRD) (E), or G245S(ΔBD) or R248W(ΔBD) (F).