XAF-1 exists as a single gene with eight exons, which localizes to chromosome 17p13.2, a region just telomeric to the p53 gene.1 Although XAF-1 has been shown to have a potent proapoptotic activity and to act as a tumor suppressor in several solid tumors by inhibiting X-linked inhibitor of apoptosis at the mitochondrial level,2 its potential role in hematological malignancies has not been addressed. As 17p- deletion has been associated with poor prognosis in patients affected by B chronic lymphocytic leukemia (B-CLL),3 we have analyzed the steady-state messenger RNA (mRNA) levels of XAF-1 in a cohort of B-CLL samples, characterized for CLL-associated chromosomal aberrations using fluorescence in situ hybridization (FISH), Zap70 levels and p53 status (Table 1). Fifteen patient samples showed a monoallelic p53 deletion associated to 17p_ (p53 deleted). Five patient samples with 17p_ also carried the p53 mutation (p53 mutated) in the remaining allele (patients 11–12; 14–16 of Table 1), while one patient sample showed p53 mutation without detectable defects on 17p at FISH analysis (patient 10 of Table 1). When assayed ex vivo for cytotoxic response to 10 mM of the non-genotoxic activator of the p53 pathway Nutlin-3,4 B-CLL samples showed a variable susceptibility to Nutlin-3 cytotoxicity upon 48 h of treatment (Table 1). Taking into consideration that patients showing p53del (17p_) and p53mut are similar in terms of poor prognosis,5 in the following analyses we have considered together patient samples with p53del and/or p53mut (p53del/mut, n¼16) versus p53wt B-CLL samples (n¼34). As expected,4 the cytotoxic activity of Nutlin-3 was greater (Po0.05) in B-CLL samples with p53wt (median cell viability of 62%, mean±s.d.: 61þ14.2) with respect to the samples with p53del/mut (median cell viability of 69%, mean±s.d.: 69.3þ13.8). In order to compare the levels of expression of XAF-1 in these two groups, the amount of mRNA was assessed using the real-time thermal analyzer Rotor- GeneTM 6000 (Corbett, Cambridge, UK) by using SYBR Green based-technology and the reverse transcriptase-polymerase chain reaction primer sets specific for the indicated human cDNA (SABioscience, Frederick, MD, USA). The steady-state XAF-1 mRNA levels were significantly (Po0.05) lower in p53del/mut (median: 2.6, meanþs.d.: 13.4þ17.5) with respect to p53wt (median: 10.3, meanþs.d.: 30.3þ36.2) B-CLL samples (Figure 1a, left panel). On the other hand, Nutlin-3 did not significantly affect XAF-1 mRNA in both p53wt and p53del/mut B-CLL cells (Figure 1a, right panel). As interferon-b (IFN-b) is a major inducer of XAF-1 in solid tumors,6 we next investigated the effect of IFN-b plus Nutlin-3 in p53wt SKW6.4 aapproximately 10-fold greater basal levels of XAF-1 mRNA with respect to BJAB (data not shown), incubation with IFN-b (500 U/ml) induced a comparable, significant increase (Po0.01) in XAF-1 mRNA steady-state levels in both cell lines (Figure 1b), and significantly (Po0.05) upregulated Nutlin-3 cytotoxicity not only in SKW6.4 and BJAB but also in primary B-CLL samples (Figure 1c). As these data suggested that increased levels of XAF-1 expression might have an important role in sensitizing leukemic cells to Nutlin-3-mediated apoptosis, in the last group of experiments XAF-1 expression was attenuated using a small interfering RNA (siRNA) transfection approach. For this purpose, SKW6.4 cells (1.25_106) were resuspended into 0.1 ml of Nucleofector solution V of the human nucleofector kit V (Amaxa, Cologne, Germany). A pool of three target-specific 20–25 nt siRNAs (human XAF1 siRNA), as well as a cocktail of three different negative control siRNAs (control siRNA), each comprising a 20–25-nt scrambled sequence that will not lead to the specific degradation of any known cellular mRNA, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The efficiency and specificity of the XAF-1 knockdown were documented in preliminary experiments by a significant decrease of the steady-state mRNA XAF-1 levels in SKW6.4 cells transfected with siRNA for XAF-1 but not in cells transfected with control scrambled siRNA (data not shown). Of note, in SKW6.4 samples in which XAF-1 expression was knocked down by transfection with XAF-1-specific siRNAs, the cytotoxicity of Nutlin-3 plus IFN-b was significantly (Po0.05) lower with respect to either cells transfected with a scrambled control siRNA or cells not transfected (Figure 1d). Our data suggest that XAF-1 might represent an important determinant in modulating the cytotoxic activity of Nutlin-3 and probably also of other pro-apoptotic drugs in B-CLL cells. In fact, p53del/mut B-CLL samples, which are less responsive to chemotherapeutic or Nutlin-3 treatment than p53wt B-CLL samples, showed low levels of XAF-1 mRNA. Moreover, modulating the steady-state mRNA levels of XAF-1 through IFN-b treatment or silencing the XAF-1 gene significantly affected the Nutlin-3-mediated cytotoxicity in selected samples of primary p53wt and p53del/mut B-CLL as well as in leukemic cell lines. Our current findings are interesting also in light of previous data showing that XAF-1 is a natural antagonist of the inhibitor of apoptosis family proteins,2 which are believed to contribute to cancer progression and to decrease the sensitivity to drug and radiation therapy. In a recent study, Steele et al.7 showed that the transcriptional blockade of p53 through pifithrin-a (PFTa) increases the Nutlin-3 ability to induce apoptosis facilitating the translocation of p53 to mitochondria. Thus, future studies should be aimed at evaluating whether permissive levels of XAF-1 are necessary for an optimal p53-mediated cytotoxic activity at the mitochondrial level. It also noted is also noteworthy that another study has shown that PFTa not only abrogates the p53 transcriptional activity, but also induces the transcriptional activity of XAF-1 expression,8 further suggesting that XAF-1 levels are an important determinant in the cytotoxic activity of p53. In conclusion, although Nutlin-3 has still not been tested in vivo in B-CLL, drugs able to increase the levels of XAF-1 should be further considered for future therapeutic combinations in the treatment of B-CLL with either Nutlin-3 or other chemotherapeutic drugs that are able to activate the p53 pathway, such as fludarabine.

The expression levels of the pro-apoptotic XAF-1 gene modulate the cytotoxic response to Nutlin-3 in B chronic lymphocytic leukemia

SECCHIERO, Paola;DI IASIO, Maria Grazia;MELLONI, Elisabetta;VOLTAN, Rebecca;Celeghini C;ZAULI, Giorgio
2010

Abstract

XAF-1 exists as a single gene with eight exons, which localizes to chromosome 17p13.2, a region just telomeric to the p53 gene.1 Although XAF-1 has been shown to have a potent proapoptotic activity and to act as a tumor suppressor in several solid tumors by inhibiting X-linked inhibitor of apoptosis at the mitochondrial level,2 its potential role in hematological malignancies has not been addressed. As 17p- deletion has been associated with poor prognosis in patients affected by B chronic lymphocytic leukemia (B-CLL),3 we have analyzed the steady-state messenger RNA (mRNA) levels of XAF-1 in a cohort of B-CLL samples, characterized for CLL-associated chromosomal aberrations using fluorescence in situ hybridization (FISH), Zap70 levels and p53 status (Table 1). Fifteen patient samples showed a monoallelic p53 deletion associated to 17p_ (p53 deleted). Five patient samples with 17p_ also carried the p53 mutation (p53 mutated) in the remaining allele (patients 11–12; 14–16 of Table 1), while one patient sample showed p53 mutation without detectable defects on 17p at FISH analysis (patient 10 of Table 1). When assayed ex vivo for cytotoxic response to 10 mM of the non-genotoxic activator of the p53 pathway Nutlin-3,4 B-CLL samples showed a variable susceptibility to Nutlin-3 cytotoxicity upon 48 h of treatment (Table 1). Taking into consideration that patients showing p53del (17p_) and p53mut are similar in terms of poor prognosis,5 in the following analyses we have considered together patient samples with p53del and/or p53mut (p53del/mut, n¼16) versus p53wt B-CLL samples (n¼34). As expected,4 the cytotoxic activity of Nutlin-3 was greater (Po0.05) in B-CLL samples with p53wt (median cell viability of 62%, mean±s.d.: 61þ14.2) with respect to the samples with p53del/mut (median cell viability of 69%, mean±s.d.: 69.3þ13.8). In order to compare the levels of expression of XAF-1 in these two groups, the amount of mRNA was assessed using the real-time thermal analyzer Rotor- GeneTM 6000 (Corbett, Cambridge, UK) by using SYBR Green based-technology and the reverse transcriptase-polymerase chain reaction primer sets specific for the indicated human cDNA (SABioscience, Frederick, MD, USA). The steady-state XAF-1 mRNA levels were significantly (Po0.05) lower in p53del/mut (median: 2.6, meanþs.d.: 13.4þ17.5) with respect to p53wt (median: 10.3, meanþs.d.: 30.3þ36.2) B-CLL samples (Figure 1a, left panel). On the other hand, Nutlin-3 did not significantly affect XAF-1 mRNA in both p53wt and p53del/mut B-CLL cells (Figure 1a, right panel). As interferon-b (IFN-b) is a major inducer of XAF-1 in solid tumors,6 we next investigated the effect of IFN-b plus Nutlin-3 in p53wt SKW6.4 aapproximately 10-fold greater basal levels of XAF-1 mRNA with respect to BJAB (data not shown), incubation with IFN-b (500 U/ml) induced a comparable, significant increase (Po0.01) in XAF-1 mRNA steady-state levels in both cell lines (Figure 1b), and significantly (Po0.05) upregulated Nutlin-3 cytotoxicity not only in SKW6.4 and BJAB but also in primary B-CLL samples (Figure 1c). As these data suggested that increased levels of XAF-1 expression might have an important role in sensitizing leukemic cells to Nutlin-3-mediated apoptosis, in the last group of experiments XAF-1 expression was attenuated using a small interfering RNA (siRNA) transfection approach. For this purpose, SKW6.4 cells (1.25_106) were resuspended into 0.1 ml of Nucleofector solution V of the human nucleofector kit V (Amaxa, Cologne, Germany). A pool of three target-specific 20–25 nt siRNAs (human XAF1 siRNA), as well as a cocktail of three different negative control siRNAs (control siRNA), each comprising a 20–25-nt scrambled sequence that will not lead to the specific degradation of any known cellular mRNA, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The efficiency and specificity of the XAF-1 knockdown were documented in preliminary experiments by a significant decrease of the steady-state mRNA XAF-1 levels in SKW6.4 cells transfected with siRNA for XAF-1 but not in cells transfected with control scrambled siRNA (data not shown). Of note, in SKW6.4 samples in which XAF-1 expression was knocked down by transfection with XAF-1-specific siRNAs, the cytotoxicity of Nutlin-3 plus IFN-b was significantly (Po0.05) lower with respect to either cells transfected with a scrambled control siRNA or cells not transfected (Figure 1d). Our data suggest that XAF-1 might represent an important determinant in modulating the cytotoxic activity of Nutlin-3 and probably also of other pro-apoptotic drugs in B-CLL cells. In fact, p53del/mut B-CLL samples, which are less responsive to chemotherapeutic or Nutlin-3 treatment than p53wt B-CLL samples, showed low levels of XAF-1 mRNA. Moreover, modulating the steady-state mRNA levels of XAF-1 through IFN-b treatment or silencing the XAF-1 gene significantly affected the Nutlin-3-mediated cytotoxicity in selected samples of primary p53wt and p53del/mut B-CLL as well as in leukemic cell lines. Our current findings are interesting also in light of previous data showing that XAF-1 is a natural antagonist of the inhibitor of apoptosis family proteins,2 which are believed to contribute to cancer progression and to decrease the sensitivity to drug and radiation therapy. In a recent study, Steele et al.7 showed that the transcriptional blockade of p53 through pifithrin-a (PFTa) increases the Nutlin-3 ability to induce apoptosis facilitating the translocation of p53 to mitochondria. Thus, future studies should be aimed at evaluating whether permissive levels of XAF-1 are necessary for an optimal p53-mediated cytotoxic activity at the mitochondrial level. It also noted is also noteworthy that another study has shown that PFTa not only abrogates the p53 transcriptional activity, but also induces the transcriptional activity of XAF-1 expression,8 further suggesting that XAF-1 levels are an important determinant in the cytotoxic activity of p53. In conclusion, although Nutlin-3 has still not been tested in vivo in B-CLL, drugs able to increase the levels of XAF-1 should be further considered for future therapeutic combinations in the treatment of B-CLL with either Nutlin-3 or other chemotherapeutic drugs that are able to activate the p53 pathway, such as fludarabine.
Secchiero, Paola; DI IASIO, Maria Grazia; Melloni, Elisabetta; Voltan, Rebecca; Celeghini, C; Tiribelli, M; Dal Bo, M; Gattei, V; Zauli, Giorgio
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11392/1392217
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