M. Saeed Sheikh

Role of Gadd45 in apoptosis.

gadd45 is a p53-regulated growth arrest and DNA-damage-inducible gene that is also regulated in a p53-independent manner. Whether Gadd45 plays a direct role in apoptosis remains unclear. Microinjection of the exogenous gadd45 expression vector into human fibroblasts has been shown to cause G2 arrest but not apoptosis. Recent studies suggest that Gadd45 may mediate genotoxic stress or Brca1-induced apoptosis via activation of c-Jun N-terminal kinase (JNK) and/or p38 mitogen-activated protein kinase (MAPK). Analyses of gadd45-deficient mice and cells have revealed that Gadd45 appears to exhibit pleiotropic effects, including cell cycle arrest at G2/M, DNA damage repair, and control of genomic stability, but is not required for radiation-induced apoptosis. Furthermore, stress-induced activation of JNK and p38 MAPK is not altered in gadd45-deficient embryonic fibroblasts, suggesting that the lack of Gadd45 may not affect the JNK and p38 MAPK activity. Thus, although the evidence from gadd45-null cells suggests that Gadd45 probably does not play a direct role in genotoxic stress-induced apoptosis, more in-depth studies are needed to firmly establish this contention.

Role of p53 family members in apoptosis.

p53‐mediated apoptosis involves multiple mechanisms. A number of p53‐regulated apoptosis‐related genes have been identified. Some of these genes encode proteins that are important in controlling the integrity of mitochondria while the others code for membrane death receptors. p53 may also induce apoptosis by interfering with the growth factor‐mediated survival signals. Although the transactivation‐deficient p53 can induce apoptosis, evidence suggests that both the transcription‐dependent and independent functions are needed for full apoptotic activity. p73 and p63 are two other members of the p53 family that show homology to p53 in their respective transactivation, DNA‐binding and oligomerization domains. Both p73 and p63 transactivate p53‐regulated promoters and induce apoptosis. Evidence suggests that both p73 and p63 may mediate apoptosis via some of the same mechanisms that are utilized by p53. However, both p73 and p63 exhibit features that are different from those of p53. Hence, both p73 and p63 are predicted to mediate apoptosis via mechanisms that are completely distinct from those engaged by p53. J. Cell. Physiol. 182:171–181, 2000. Published 2000 Wiley‐Liss, Inc.

TC21 mediates transformation and cell survival via activation of phosphatidylinositol 3-kinase/Akt and NF-κB signaling pathway

The signaling pathways of TC21-mediated transformation and cell survival are not well-established. In this study, we have investigated the role of PI3-K/Akt signaling pathway in oncogenic-TC21-mediated transformation and cell survival. We found that oncogenic-TC21 stimulated the PI3-K activity. This was associated with the activation of Akt, a key component of PI3-K signaling pathway. We also found that TC21 interacted and formed complex with PI3-K. Mutations in the GTP-binding region of TC21, which enhanced GTP-binding potential of this protein, also stimulated its association with PI3-K, suggesting that PI3-K may preferentially interact with the GTP-bound form. Suppression of PI3-K and Akt by specific inhibitors LY294002 and Wortmannin reversed TC21-induced transformation. Likewise, inhibition of PI3-K activity by the PI3-K phosphotase PTEN reduced TC21-mediated focus formation in NIH3T3 cells. Investigation of TC21s effect on cell survival revealed that mutant-TC21 expressing cells were more resistant to etoposide- and cisplatin-induced cell death, and this was associated with the activation of anti-apoptotic protein NF-κB, a downstream target of Akt. Treatment of PI3-K inhibitor LY294002 significantly suppressed TC21-mediated NF-κB activation. In conclusion, we have identified PI3-K as an effector of TC21 and demonstrated that the PI3-K/Akt signaling pathway plays important roles in TC21-mediated transformation and cell survival.

The antiapoptotic decoy receptor TRID/TRAIL-R3 is a p53-regulated DNA damage-inducible gene that is overexpressed in primary tumors of the gastrointestinal tract.

Both DR4 and DR5 have recently been identified as membrane death receptors that are activated by their ligand TRAIL to engage the intracellular apoptotic machinery. TRID (also named as TRAIL-R3) is an antagonist decoy receptor and lacks the cytoplasmic death domain. TRID protects from TRAIL-induced apoptosis by competing with DR4 and DR5 for binding to TRAIL. TRID has been shown to be overexpressed in normal human tissues but not in malignantly transformed cell lines. DR5 is a p53-regulated gene and we have recently reported that DR5 expression is induced in response to genotoxic stress in both a p53-dependent and independent manner (Sheikh et al., 1998). In the current study, we demonstrate that TRID gene expression is also induced by the genotoxic agents ionizing radiation and methyl methanesulfonate (MMS) in predominantly p53 wild-type cells, whereas UV-irradiation does not induce TRID gene expression. Consistent with these results, exogenous wild-type p53 also upregulates the expression of endogenous TRID in p53-null cells. Thus, TRID appears to be a p53 target gene that is regulated by genotoxic stress in a p53-dependent manner. Using primary gastrointestinal tract (GIT) tumors and their matching normal tissue, we also demonstrate for the first time that TRID expression is enhanced in primary tumors of the GIT. It is, therefore, possible that TRID overexpressing GIT tumors may gain a selective growth advantage by escaping from TRAIL-induced apoptosis.

Ultraviolet-irradiation-induced apoptosis is mediated via ligand independent activation of tumor necrosis factor receptor 1

Ultraviolet (UV)-irradiation has been shown to induce jun N-terminal kinase activity via aggregation-mediated activation of tumor necrosis factor receptor 1 (TNFR1) but the role of TNFR1 in mediating UV-induced apoptosis has not been explored. Using p53-null cells, we demonstrate that UV-stimulated ligand independent activation of TNFR1 plays a major role in mediating the apoptotic effects of UV-irradiation. UV-irradiation and TNFα acted in a synergistic manner to induce apoptosis. UV-irradiation stimulated the aggregation-mediated activation of TNFR1 which was coupled with activation of caspase 8, the most proximal caspase in TNFα signaling pathway. CrmA and the dominant negative versions of FADD, caspase 8 and caspase 10, that block the apoptotic axis of TNFR1 at different levels, also independently inhibited the UV-induced apoptosis. The engagement of the membrane initiated events was specific for UV-irradiation since neither CrmA nor the dominant negative FADD, caspase 8 or caspase 10 blocked the ionizing radiation-induced apoptosis. Cisplatin and melphalan, the UV-mimetic agents known to elicit UV-type DNA damage, also induced apoptosis but differed from UV in that both of the former agents engaged the caspase cascade at a level distal to FADD. Consistent with these findings cisplatin also did not stimulate TNFR1 aggregation. Together these results indicate that DNA damage per se was not sufficient to activate the membrane TNFR1. Based on our results we propose that the plasma membrane initiated events play a predominant role in mediating UV-irradiation-induced apoptosis and that UV-irradiation appears to engage the apoptotic axis of TNFR1 and perhaps those of other membrane death receptors to transduce its apoptotic signals.

Uniform nomenclature for the mitochondrial contact site and cristae organizing system

The mitochondrial inner membrane contains a large protein complex that functions in inner membrane organization and formation of membrane contact sites. The complex was variably named the mitochondrial contact site complex, mitochondrial inner membrane organizing system, mitochondrial organizing structure, or Mitofilin/Fcj1 complex. To facilitate future studies, we propose to unify the nomenclature and term the complex “mitochondrial contact site and cristae organizing system” and its subunits Mic10 to Mic60.

Role of p21Waf1/Cip1/Sdi1 in cell death and DNA repair as studied using a tetracycline-inducible system in p53-deficient cells

Postulated roles for p21Waf1/Cip1/Sdi1 (p21) in DNA repair and apoptosis remain controversial. Studies suggest both stimulatory and inhibitory effects of p21 in DNA repair. p21 has also been implicated in induction or protection from apoptosis. Using the tetracycline inducible expression system, we studied the role of p21 in DNA repair and apoptosis in wild-type p53 deficient DLD1 colorectal carcinoma cells. These cells displayed marked heterogeneity in their ability to tolerate higher levels of exogenous p21. The majority of the p21 overexpressing cells grew slower and did not exhibit apoptotic phenotype, some cells underwent apoptotic death within 5 – 8 days following p21 induction while other became giant cells prior to undergoing cell death. Induction of p21 transgene neither sensitized to nor protected from adriamycin-induced acute cell death. p21 also did not alter the clonogenic survival following adriamycin treatment. Clonogenic survival after u.v.-irradiation was, however, increased when p21 expression was transiently induced a few hours before and after u.v.-irradiation. Consistent with its effect on clonogenic survival, p21 also enhanced the cellular capacity to repair three different exogenously introduced u.v.-damaged reporter plasmids. Taken together our results demonstrate that p21 may modulate the nucleotide excision repair process to facilitate the repair of u.v.-type DNA damage even in the absence of wild-type p53.

Proteasome inhibitor MG132 upregulates death receptor 5 and cooperates with Apo2L/TRAIL to induce apoptosis in Bax-proficient and -deficient cells

Apo2L/TRAIL (tumor necrosis factor-related apoptosis inducing ligand (TRAIL), also known as Apo2L) is a potentially important anticancer agent awaiting clinical trials. Unfortunately, however, some cancer cells exhibit resistance to Apo2L/TRAIL, which could limit the use of this potentially promising anticancer agent. Although the molecular basis of the inherent or acquired resistance to Apo2L/TRAIL remains unclear, previous studies indicate that Bax deficiency can confer resistance to Apo2L/TRAIL. Proteasome inhibition is also emerging as a promising therapeutic strategy to manage human malignancies. Here, we report that proteasome inhibitor MG132 upregulates Apo2L/TRAIL death receptor 5 expression in both Bax-proficient and -deficient HCT116 cells. MG132 effectively cooperated with Apo2L/TRAIL to induce apoptosis in both Bax-proficient and -deficient cells that was coupled with caspases-8 and -3 activation and Bid cleavage. Although both agents in combination also induced cytochrome c and Smac release from mitochondria into cytosol and activated caspase-9 in Bax-proficient cells, their effects on these events were significantly diminished in Bax-deficient cells. These results suggest that Bax is not absolutely required for death receptor 5-dependent apoptotic signals and MG132 by upregulating DR5 effectively cooperates with Apo2L/TRAIL to overcome Bax deficiency-induced resistance to Apo2L/TRAIL. Our results have important clinical implications in that the use of Apo2L/TRAIL and proteasome inhibitors in combination could prove to be a novel therapeutic strategy to manage the Apo2L/TRAIL-resistant tumors.

Regulation of translation initiation following stress

Recent studies suggest that genotoxic and non-genotoxic stresses appear to invoke translational checkpoints in order to inhibit protein synthesis. Depending on the stress and/or cell type, this downregulation of protein synthesis may either (i) protect against the deleterious effects of noxious agents and ensure the conservation of resources that are needed to survive under adverse conditions or (ii) activate apoptosis. In this article, we have reviewed several lines of evidence which support the notion that regulation of translation initiation is an important component of the cellular stress response. While the stress-induced post-translational regulation of translation initiation factors (eIFs) has been well documented, stress-induced regulation of eIFs at the mRNA levels, as reviewed here, is only beginning to be elucidated. Thus, the stress-mediated regulation of eIFs occurs at multiple different levels involving, transcriptional, post-transcriptional and post-translational controls.

Tumor suppressor RASSF1A is a microtubule-binding protein that stabilizes microtubules and induces G2/M arrest

RASSF1A is a putative tumor suppressor gene that is inactivated in a variety of human tumors. Expression of exogenous RASSF1A has been shown to inhibit tumor growth in vitro and in animals. However, the molecular mechanisms by which RASSF1A mediates its tumor suppressive effects remain to be elucidated. Here, we report that RASSF1A is a microtubule-binding protein that interacts with and stabilizes microtubules. We have identified the RASSF1A region harboring a basic domain that appears to mediate the interactions between RASSF1A and microtubules. The basic domain-containing RASSF1C isoform also interacts with and stabilizes microtubules. We further show that in addition to G1 arrest, RASSF1A promotes growth arrest in the G2/M phase of the cell cycle and endogenous RASSF1A also interacts with microtubules. Based on our results, we propose that RASSF1A may mediate its tumor suppressive effects by inducing growth arrest in the G1 and G2/M phases. Together, these results provide important new insights into the molecular mechanisms by which this novel tumor suppressor mediates its biological effects.