For this study, primary astrocytes were generated from Ink4a/Arf-/-PTEN+/+ or Ink4a/Arf-/-PTENf/f transgenic littermates. Once in culture, floxed PTEN alleles were deleted by adenoviral expression of Cre recombinase, generating a set of matched astrocytes with the following genotypes: Ink4a/Arf-/-PTEN+/+ and Ink4a/Arf-/-PTEN-/-. Because primary mouse astrocytes senesce within a couple of passages after extraction, the Ink4a/Arf-/- background ensures that the astrocytes are immortal and provides a tumor-suppressor background that is very relevant to GBMs (5
). PTEN deletion upon adenovirus infection was confirmed by Western blotting (), as well as by PCR analysis (Supplement; Fig. S1
). As expected, PTEN loss strongly activated PI3K signaling as evidenced by increased levels of phosphorylated Akt-1 (phospho-serine 473) (11
). In accordance with previous reports (22
), we found that loss of PTEN resulted in increased resistance to IR as assayed by colony survival (). In contrast, PTEN loss resulted in sensitization to MNNG (). Flow cytometric analyses revealed a significant increase in the sub-G0 population in MNNG-treated PTEN-deficient cultures indicating that the sensitivity of these astrocytes to MNNG was due to an increase in cell death ().
PTEN loss sensitizes astrocytes to MNNG
While the cytoplasmic role of PTEN in squelching the PI3K-Akt-1 pathway is an established concept, recent reports clearly indicate novel nuclear functions for this protein, including roles in transcription regulation (11
). The sensitivity of PTEN-null cells to MNNG was probably not due to hyperactivation of Akt-1 as astrocytes expressing constitutively active myristylated Akt-1 (8
) did not display an increased sensitivity to MNNG compared to parental cells (Supplement; Fig. S2
). Therefore, it is plausible that the sensitivity to MNNG observed in PTEN-/- astrocytes was due to loss of a nuclear function of PTEN.
Toxicity from SN
1-type alkylating agents results mainly from a specific type of DNA lesion, methylation of the O6
position of guanine (O6
), that can be reversed by the suicide repair enzyme, MGMT (25
). It has been previously reported that MGMT
promoter silencing by methylation corresponds to a better therapeutic response to temozolomide (3
). Therefore, we investigated whether a decrease in MGMT levels due to PTEN loss might underlie the sensitivity of these cell lines to MNNG. However, Western blot analyses of basal MGMT protein levels did not show a significant difference between PTEN+/+ and PTEN-/- astrocytes (). MGMT transcription is induced in response to various DNA damaging agents, including MNNG (24
). However, quantitative real-time PCR (qRT-PCR) analyses revealed that both lines were capable of inducing MGMT transcription upon MNNG treatment (). These data strongly suggest that the sensitivity of PTEN-null cells to MNNG was not due to attenuation of MNNG
transcript or protein levels.
MGMT regulation is not affected by PTEN loss
The cytotoxicity of O6
meG lesions is attributed to the recognition of O6
-meG/C or O6
-meG/T mispairs by the mismatch repair (MMR) system (27
), with two opposing models proposed: (a) DNA damage signal transduction by the MMR complex engaged at the mismatch sites directly triggering apoptosis (direct signaling model) or (b) reiterative and futile repair attempts by MMR resulting in single-strand and double-strand DNA breaks (futile cycle model) (28
). Since DNA double-strand breaks (DSBs) are the most lethal of DNA lesions, we investigated whether such breaks were induced in MNNG-treated astrocytes and whether these breaks were more persistent in PTEN-deficient cells. We analyzed the formation and dissolution of γH2AX and 53BP1 foci upon pulse-treatment with MNNG (8
), these foci being bona fide
surrogate markers for DSBs (30
). Interestingly, MNNG induced equivalent levels of DSBs in both lines; however, PTEN-deficient astrocytes exhibited higher levels of DSBs at 24 h post-treatment indicating a deficiency in repair of these breaks ().
PTEN loss compromises homologous recombination repair
DSBs are repaired by NHEJ or HR in mammalian cells. While NHEJ is operative in all phases of the cell cycle, HR is limited to S/G2 and is particularly important for resolving replication-associated breaks (32
). MNNG-induced breaks are presumed to occur in the S/G2 phases as DNA replication is required for mispairing, and this is borne out by our observations indicating that MNNG-induced H2AX phosphorylation occurs only in S/G2 cells (Supplement; Fig. S3
). Therefore, it is likely that these breaks may be resolved by HR rather than by NHEJ. Indeed, we observed no further sensitization upon treating these cells with NU7026, a potent inhibitor of the major NHEJ repair enzyme, DNA-PKcs (33
) (Supplement; Fig. S4
), thereby implicating HR in repair. In support of this idea, a recent report demonstrated that MNNG induces DSBs and that cells defective in HR (XRCC2 and Brca2 mutants), but not cells defective in NHEJ (Ku80 and DNA-PKcs mutants), were sensitive to MNNG, similar to our PTEN-null cells (34
Cells deficient in various HR components show a decrease in the number of sister chromatid exchanges (SCEs) after treatment with DNA damaging agents (35
), especially agents that induce replication-associated DSBs such as camptothecin (CPT) (36
). Also, HR-deficient cells are sensitive to CPT (36
) and we found that PTEN-deficient astrocytes were more sensitive to this drug compared to their PTEN-proficient counterparts (). We quantified the number of SCEs in PTEN+/+ and PTEN-/- astrocytes after treatment with CPT or MNNG to determine relative HR proficiencies of these lines. A statistically significant reduction in SCE events was observed in PTEN-/- astrocytes relative to PTEN+/+ astrocytes indicating a defect in HR (). Consequently, PTEN-null cells surviving MNNG treatment exhibited greater numbers of chromosome breaks and radial chromosomes (), similar to that seen in HR-deficient cells, particularly those deficient in Brca1 or Brca2 (37
). These aberrations are indicative of a diminished capacity to repair MNNG-induced DSBs by error-free HR and subsequent repair of these lesions by error-prone pathways such as NHEJ.
Interestingly, Shen et al. recently demonstrated that PTEN is important for maintaining basal levels of transcription of the Rad51
gene in mouse embryonic fibroblasts (12
), providing a potential mechanism to explain the reduced HR capability of PTEN-null astrocytes. However, no significant changes in Rad51 protein or mRNA levels in mouse astrocytes upon PTEN loss were noted (). While Rad51 forms a presynaptic nucleofilament that is critical for HR, ancillary proteins such as BRCA1, BRCA2, Rad52, and the Rad51 paralogs (Rad51B, Rad51C, Rad51D, XRCC2, and XRCC3) facilitate multiple steps during the repair process (38
). Because there are numerous recent reports of PTEN acting as a transcriptional regulator (13
), we screened several of these “recombination mediators” for expression changes upon PTEN loss by qRT-PCR and observed decreases in the transcript levels of Rad51B, C, and D (). As these proteins are known to exist in complexes facilitating Rad51 nucleofilament formation (38
), it is plausible that reduced levels of these proteins could result in attenuated HR upon PTEN loss.
PTEN-null astrocytes express lower levels of Rad51 paralogs and are sensitive to PARP inhibitors
A very important prediction from the observed deficiency in HR is that PTEN-null astrocytes should be sensitive to PARP inhibitors that induce replication-associated DSBs. This phenomenon of “synthetic lethality” was originally identified in the context of BRCA1 and BRCA2 mutations in breast cancer (14
), and PARP inhibitors are now in clinical trials for treating HR-deficient breast and ovarian cancers (16
). We found that PTEN-null cells were significantly more sensitive to the PARP inhibitor, ABT-888 (39
), compared to PTEN-proficient cells (). The sensitivity to ABT-888 is consistent with a HR-deficiency in PTEN-null cells, and suggests that it might be logical to treat PTEN-deficient GBMs with PARP inhibitors in the future.
The isogenic murine astrocytes used in this study are ideal for analyzing the effect of a single genetic change (PTEN loss) on MNNG sensitivity. However, in the context of human GBMs, the effect of a single genetic lesion could be modulated by innumerable background genetic changes (5
). To explore the relevance of our findings in human GBMs, we compared two commonly used glioma lines (U87MG and U251MG) with a normal human astrocyte line (NHA) that had been immortalized by expression of human telomerase catalytic component (hTERT) and human papillomavirus 16 E6/E7 proteins (40
). Both glioma lines are PTEN-null (41
) and were more sensitive to MNNG compared to the NHA line, which has an intact PTEN
gene (Supplement; Fig. S5
). These results tentatively suggest that PTEN-deficient glioblastoma cells may be more sensitive to DNA alkylating agents compared to PTEN-proficient normal human astrocytes and this could confer a selective advantage to DNA alkylating agents for the treatment of PTEN-null GBMs. Importantly, siRNA-mediated depletion of PTEN rendered the NHA line sensitive to MNNG as quantified by the colony formation assay (Supplement; Fig. S6
). This was possibly due to attenuated HR, as we observed a reduced induction of SCEs upon PTEN depletion. Interestingly, SCE induction in PTEN-null U87 cells were also reduced compared to the PTEN-proficient NHA line. More importantly, PTEN depletion could also sensitize transformed, gliomagenic NHAs (expressing E6, E7, hTERT, H-Ras, and myristylated Akt-1) (42
), indicating that PTEN loss might result in sensitivity to DNA alkylating agents in the context of human gliomas. In sum, these results confirm that, as observed in murine astrocytes, PTEN loss plays an important role in modulating MNNG sensitivity of normal human astrocytes and gliomagenic derivatives.