Philipp Hemmati

Adenovirus-mediated overexpression of p14(ARF) induces p53 and Bax-independent apoptosis.

The human INK4a gene locus encodes two structurally unrelated tumor suppressor proteins, p16INK4a and p14ARF, which are frequently inactivated in human cancer. Whereas p16INK4a acts through engagement of the Rb-cdk4/6-cyclin D pathway, both the pro-apoptotic and cell cycle-regulatory functions of p14ARF were shown to be primarily dependent on the presence of functional p53. Recent reports have also implicated p14ARF in p53-independent mechanisms of cell cycle regulation and apoptosis induction, respectively. To further explore the pro-apoptotic function of p14ARF in relation to functional cellular p53, we constructed a replication-deficient adenoviral vector for overexpression of p14ARF (Ad-p14ARF). As expected, Ad-p14ARF efficiently induced apoptosis in p53/Rb wild-type U-2OS osteosarcoma cells at low multiplicities of infection. Interestingly, Ad-p14ARF also induced apoptosis in both p53-deleted SAOS-2 osteosarcoma cells and HCT116 colon cancer cells with a bi-allelic knock-out of p53 (HCT116-p53−/−). Similarly, adenovirus-mediated overexpression of p14ARF induced apoptosis in p53/Bax-mutated DU145 prostate cancer cells as well as in HCT116 cells devoid of functional Bax (HCT116-Bax−/−). Restoration of Bax expression by retroviral gene transfer in DU145 cells did not further enhance p14ARF-triggered cell death. Infection with Ad-p14ARF induced activation of mitochondrial permeability shift transition, caspase activation and apoptotic DNA fragmentation irrespective of the presence or absence of either Bax or functional cellular p53. Nevertheless, overexpression of the anti-apoptotic Bcl-2 homolog Bcl-xL markedly inhibited p14ARF-induced apoptosis. This may indicate that p14ARF triggers a so far unknown activator of mitochondrial apoptosis which can be inhibited by Bcl-2 but which acts either independently or downstream of Bax. Taken together, this report demonstrates the participation of signaling pathways apart from the p53/Mdm-2 rheostat and Bax in p14ARF-mediated apoptosis.

Multidomain Bcl-2 homolog Bax but not Bak mediates synergistic induction of apoptosis by TRAIL and 5-FU through the mitochondrial apoptosis pathway

The death ligand TRAIL synergizes with DNA-damaging therapies such as chemotherapeutic drugs or ionizing irradiation. Here, we show that the synergism of TRAIL and 5-fluorouracil (5-FU) and cross-sensitization between TRAIL and 5-FU for induction of apoptosis, entirely depend on Bax proficiency in human DU145 and HCT116 carcinoma cells. DU145 prostate carcinoma cells that have lost Bax protein expression due to mutation fail to release cytochrome c and to activate caspase-3 and -9 when exposed to TRAIL and 5-FU. In contrast, TRAIL sensitized for 5-FU-induced apoptosis and vice versa upon reconstitution of Bax expression. Isobolographic analyses of ED50 doses for 5-FU at increasing TRAIL concentrations showed a clear synergism of TRAIL and 5-FU in Bax-expressing cells. In contrast, the effect was merely additive in DU145 cells lacking Bax. Notably, both DU145 and HCT116 Bax-deficient cells still express Bak. This indicates that Bak is not sufficient to mediate cross-sensitization and synergism between 5-FU and TRAIL. Stable overexpression of Bak in DU145 sensitized for epirubicin-induced apoptosis but failed to confer synergy between TRAIL and 5-FU. Moreover, we show by the use of EGFP-tagged Bax and Bak that TRAIL and 5-FU synergistically trigger oligomerization and clustering of Bax but not Bak. These data clearly establish distinct roles for Bax and Bak in linking the TRAIL death receptor pathway to the mitochondrial apoptosis signaling cascade and delineate a higher degree of specificity in signaling for cell death by multidomain Bcl-2 homologs.

Induction of p21CIP/WAF-1 and G2 arrest by ionizing irradiation impedes caspase-3-mediated apoptosis in human carcinoma cells

There is an ongoing controversy regarding the relevance of apoptosis induction by ionizing irradiation as compared with other end points including transient or permanent cell cycle arrest of damaged cells. Here, we show that such permanent cell cycle arrest and apoptosis represent two sides of the same coin. MCF-7 cells fail to express procaspase-3, which results in resistance to apoptosis induced by anticancer drugs. Conversely, restoration of procaspase-3 sensitizes MCF-7 cells to chemotherapeutics including epirubicine, etoposide and taxol. In contrast, irradiation does not trigger apoptotic cell death but results in prolonged arrest in the G2 phase of the cell division cycle regardless of procaspase-3 expression. This suggested that the propensity of MCF-7 cells to arrest at the G2 checkpoint results in resistance to apoptosis upon γ-irradiation. This G2 arrest was associated with upregulation of p21CIP/WAF-1. Inhibition of DNA-damage-induced stress kinases and p21CIP/WAF-1 expression by caffeine abrogated G2 arrest and induced apoptosis of the irradiated cells in a caspase-3-dependent manner. Inhibition of cell cycle progression by adenoviral expression of the cyclin dependent kinase inhibitor p21CIP/WAF-1 prevented apoptosis upon caffeine treatment indicating that cell cycle progression, that is, G2-release, is required for induction of apoptosis. Likewise, cells homozygously deleted for p21CIP/WAF-1 (HCT116 p21−/−) display enhanced irradiation-induced apoptosis via a caspase-3-dependent mechanism. These data indicate that the disruption of G2 checkpoint control overcomes cell cycle arrest and resistance to γ-irradiation-induced cell death. Thus, DNA damage may trigger a permanent G2 arrest as an initial inactivation step of tumor cells where the phenomenon of apoptosis is hidden unless cell cycle arrest is overcome. The efficient induction of apoptosis upon G2 release thereby depends on the propensity to activate the key executioner caspase-3. This finding is of crucial importance for the understanding of molecular steps underlying the efficacy of ionizing radiation to delete tumor cells.

Smac induces cytochrome c release and apoptosis independently from Bax/Bcl-xL in a strictly caspase-3-dependent manner in human carcinoma cells

The mitochondrial apoptosis pathway mediates cell death through the release of various pro-apoptotic factors including cytochrome c and Smac, the second mitochondrial activator of caspases, into the cytosol. Smac was shown previously to inhibit IAP proteins and to facilitate initiation of the caspase cascade upon cytochrome c release. To investigate Smac function during apoptosis and to explore Smac as an experimental cancer therapeutic, we constructed an expression system based on a single adenoviral vector containing Smac under control of the Tet-off system supplied in cis. Conditional expression of Smac induced apoptosis in human HCT116 and DU145 carcinoma cells regardless of the loss of Bax or overexpression of Bcl-xL. Nevertheless, apoptosis induced by Smac was associated with cytochrome c release and breakdown of the mitochondrial membrane potential. This indicates that Smac acts independently of Bax and Bcl-xL during initiation of apoptosis and triggers a positive feedback loop that results in Bax/Bcl-xL-independent activation of mitochondria. In caspase-proficient cells, Smac-induced apoptosis could be inhibited partially by cell-permeable LEHD (caspase-9 inhibitor) and DEVD (caspase-3 inhibitor) peptides. Furthermore, loss of caspase-3 expression in MCF-7 cells carrying a caspase-3 null mutation completely abrogated the sensitivity for Smac-induced apoptotic or nonapoptotic, necrosis-like cell death, while re-expression of caspase-3 conferred sensitivity. Altogether, caspase-3 but not caspase-9 activation was necessary for execution of Smac-induced cell death. Notably, Smac did not induce caspase-9 processing in the absence of caspase-3. Thus, caspase-9 processing occurs secondary to caspase-3 activation during Smac-induced apoptosis. Altogether, Smac is capable of circumventing defects in mitochondrial apoptosis signaling such as loss of Bax or overexpression of Bcl-xL that are frequently observed in tumor cells resistant to anticancer therapy. Consequently, Smac appears to be a promising therapeutic target in anticancer treatment.

TRAIL sensitizes for ionizing irradiation-induced apoptosis through an entirely Bax-dependent mitochondrial cell death pathway.

The death ligand TRAIL has been suggested as a suitable biological agent for the selective induction of cell death in cancer cells. Moreover, TRAIL synergizes with DNA-damaging therapies such as chemotherapeutic drugs or ionizing irradiation (IR). Here, we show that synergy of TRAIL and IR, that is, crosssensitization between TRAIL and IR for induction of apoptosis, entirely depends on Bax proficiency in human DU145 and HCT116 carcinoma cells. DU145 prostate carcinoma cells that have lost Bax protein expression due to mutation fail to activate caspase-3 and -9 when exposed to TRAIL and IR. In contrast, TRAIL sensitized for IR-induced apoptosis and vice versa upon reconstitution of Bax expression. Notably, both DU145 and HCT116 still express significant levels of the multidomain proapoptotic Bcl-2 homolog Bak. This indicates that Bak is not sufficient to mediate crosssensitization and synergism between IR and TRAIL. These data clearly establish distinct roles for Bax and Bak in linking the TRAIL death receptor pathway to the mitochondrial apoptosis signaling cascade upon DNA damage by IR.

Bone marrow T-cell infiltration during acute GVHD is associated with delayed B-cell recovery and function after HSCT

B-cell immune dysfunction contributes to the risk of severe infections after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Delayed B-cell regeneration is found in patients with systemic graft-versus-host disease (GVHD) and is often accompanied by bone marrow (BM) suppression. Little is known about human BM GVHD. We analyzed the reconstitution kinetics of B-cell subsets in adult leukemic patients within 6 months after allo-HSCT. B-cell deficiency already existed before transplant and was aggravated after transplant. Onset of B-cell reconstitution characterized by transitional B-cell recovery occurred either early (months 2-3) or late (from month 6 on) and correlated highly positively with reverse transcription-polymerase chain reaction quantified numbers of κ-deleting recombination excision circles (KRECs). Delayed recovery was associated with systemic acute GVHD and full-intensity conditioning therapy. Histological analysis of BM trephines revealed increased T-cell infiltration in late recovering patients, which was associated with reduced numbers of osteoblasts. Functionally, late recovering patients displayed less pneumococcal polysaccharide-specific immunoglobin M-producing B cells on ex vivo B-cell activation than early recovering patients. Our results provide evidence for acute BM GVHD in allo-HSCT patients with infiltrating donor T cells and osteoblast destruction. This is associated with delayed B-cell reconstitution and impaired antibody response. Herein, KREC appears suitable to monitor BM B-cell output after transplant.

A modified EBMT risk score and the hematopoietic cell transplantation-specific comorbidity index for pre-transplant risk assessment in adult acute lymphoblastic leukemia

Background Disease stage is the most important prognostic parameter in allogeneic hematopoietic cell transplantation (HCT) for acute lymphoblastic leukemia, but other factors such as donor/host histocompatibility and gender combination, recipient age, performance status and comorbidities need to be considered. Several scoring systems are available to predict outcome in HCT recipients; however, their prognostic relevance in acute lymphoblastic leukemia is not well defined. Design and Methods In the present study we evaluated a modified EBMT risk score (mEBMT) and the HCT-specific comorbidity index (HCT-CI) in 151 adult acute lymphoblastic leukemia patients who received allogeneic HCT from 1995 until 2007 at our center. Results Disease status was first complete remission (CR1) (47%), CR>1 (21%) or no CR (32%). Overall survival (OS) at one, two and five years was 62%, 51% and 40% and non-relapse mortality (NRM) was 21%, 24% and 32%. Median mEBMT was 3 (0–6). Higher mEBMT was associated with inferior OS (hazard ratio per score unit (HR): 1.50, P<0.001), higher NRM (HR: 1.36, P=0.042) and higher relapse mortality (HR: 1.68, P<0.001). Disease stage was the predominant prognostic factor in this score. Comorbidities were present in 71% of patients with mild hepatic disease (29%), moderate pulmonary disease (28%) and infections (23%) being the most common. Median HCT-CI was 1 (0–9). In univariate analysis a trend for inferior OS (HR: 1.08, P=0.20) and higher NRM (HR: 1.14, P=0.11) with increasing HCT-CI was observed but the level of significance was not reached. In additional analyses we found that reduced Karnofsky Performance Status (KPS) was associated with inferior OS (HR: 1.34, P=0.023) and higher relapse mortality (HR: 1.71, P=0.001) when analyzed univariately. However, KPS was associated with disease stage and significance was lost in multivariate analysis. Conclusions The mEBMT was prognostic in our patient cohort with predominant influence of disease stage, whereas a trend but no significant prognostic value was observed for the HCT-CI.

Mcl-1 determines the Bax dependency of Nbk/Bik-induced apoptosis

B cell lymphoma 2 (Bcl-2) homology domain 3 (BH3)–only proteins of the Bcl-2 family are important functional adaptors that link cell death signals to the activation of Bax and/or Bak. The BH3-only protein Nbk/Bik induces cell death via an entirely Bax-dependent/Bak-independent mechanism. In contrast, cell death induced by the short splice variant of Bcl-x depends on Bak but not Bax. This indicates that Bak is functional but fails to become activated by Nbk. Here, we show that binding of myeloid cell leukemia 1 (Mcl-1) to Bak persists after Nbk expression and inhibits Nbk-induced apoptosis in Bax-deficient cells. In contrast, the BH3-only protein Puma disrupts Mcl-1–Bak interaction and triggers cell death via both Bax and Bak. Targeted knockdown of Mcl-1 overcomes inhibition of Bak and allows for Bak activation by Nbk. Thus, Nbk is held in check by Mcl-1 that interferes with activation of Bak. The finding that different BH3-only proteins rely specifically on Bax, Bak, or both has important implications for the design of anticancer drugs targeting Bcl-2.

p14ARF Induces G2 Cell Cycle Arrest in p53- and p21-deficient Cells by Down-regulating p34cdc2 Kinase Activity

The human INK4a gene locus encodes two structurally unrelated tumor suppressor proteins, p16INK4a and p14ARF. Although primarily proposed to require a functional p53·Mdm-2 signaling axis, recently p14ARF has been implicated in p53-independent cell cycle regulation. Here we show that p14ARF preferentially induces a G2 arrest in tumor cells lacking functional p53 and/or p21. Expression of p14ARF impaired mitotic entry and enforced a primarily cytoplasmic localization of p34cdc2 that was associated with a decrease in p34cdc2 kinase activity and reduced p34cdc2 protein expression. A direct physical interaction between p14ARF and p34cdc2 was, nevertheless, ruled out by lack of co-immunoprecipitation. The p14ARF-induced depletion of p34cdc2 was associated with impaired cdc25C phosphatase expression and a prominent shift to inhibitory Tyr-15-phosphorylation in G2-arrested cells lacking either p53, p21, or both. Finally, reconstitution of p34cdc2 using a constitutively active, phosphorylation-deficient p34cdc2AF mutant alleviated this p14ARF-induced G2 arrest, thereby allowing cell cycle progression. Taken together, these data indicate that p14ARF arrests cells lacking functional p53/p21 in the G2 phase of the cell cycle by targeting p34cdc2 kinase. This may represent an important fail-safe mechanism by which p14ARF protects p53/p21-deficient cells from unrestrained proliferation.

Bak functionally complements for loss of Bax during p14ARF-induced mitochondrial apoptosis in human cancer cells.

In contrast to the initial notion that the biological activity of p14ARF strictly depends on a functional mdm-2/p53 signaling axis, we recently demonstrated that p14ARF mediates apoptosis in a p53/Bax-independent manner. Here, we show that p14ARF induces breakdown of the mitochondrial membrane potential and cytochrome c release before triggering caspase-9- and caspase-3/7-like activities in p53/Bax-deficient DU145 prostate cancer cells expressing wild-type Bak. Re-expression of Bax in these cells failed to further enhance p14ARF-induced apoptosis, suggesting that p14ARF-induced apoptosis primarily depends on Bak but not Bax in these cells. To further define the role of Bak and Bax in p14ARF-induced mitochondrial apoptosis, we employed short interference RNA for the knockdown of bak in isogeneic, p53 wild-type HCT116 colon cancer cells either proficient or deficient for Bax. There, combined loss of Bax and Bak attenuated p14ARF-induced apoptosis whereas single loss of Bax or Bak was only marginally effective, as in the case of DU145. Notably, HCT116 cells deficient for Bax and Bak failed to release cytochrome c and showed attenuated activation of caspase-9 (LEHDase) and caspase-3/caspase-7 (DEVDase) upon p14ARF expression. These data indicate that p14ARF triggers apoptosis via a Bax/Bak-dependent pathway in p53-proficient HCT116, whereas Bax is dispensable in p53-deficient DU145 cells. Nevertheless, a substantial proportion of p14ARF-induced cell death proceeds in a Bax/Bak-independent manner. This is also the case for inhibition of clonogenic growth that occurs, at least in part, through an entirely Bax/Bak-independent mechanism.