Vijaya Chaturvedi

Proteasome inhibitors trigger NOXA-mediated apoptosis in melanoma and myeloma cells

Patients with metastatic melanoma or multiple myeloma have a dismal prognosis because these aggressive malignancies resist conventional treatment. A promising new oncologic approach uses molecularly targeted therapeutics that overcomes apoptotic resistance and, at the same time, achieves tumor selectivity. The unexpected selectivity of proteasome inhibition for inducing apoptosis in cancer cells, but not in normal cells, prompted us to define the mechanism of action for this class of drugs, including Food and Drug Administration-approved bortezomib. In this report, five melanoma cell lines and a myeloma cell line are treated with three different proteasome inhibitors (MG-132, lactacystin, and bortezomib), and the mechanism underlying the apoptotic pathway is defined. Following exposure to proteasome inhibitors, effective killing of human melanoma and myeloma cells, but not of normal proliferating melanocytes, was shown to involve p53-independent induction of the BH3-only protein NOXA. Induction of NOXA at the protein level was preceded by enhanced transcription of NOXA mRNA. Engagement of mitochondrial-based apoptotic pathway involved release of cytochrome c, second mitochondria-derived activator of caspases, and apoptosis-inducing factor, accompanied by a proteolytic cascade with processing of caspases 9, 3, and 8 and poly(ADP)-ribose polymerase. Blocking NOXA induction using an antisense (but not control) oligonucleotide reduced the apoptotic response by 30% to 50%, indicating a NOXA-dependent component in the overall killing of melanoma cells. These results provide a novel mechanism for overcoming the apoptotic resistance of tumor cells, and validate agents triggering NOXA induction as potential selective cancer therapeutics for life-threatening malignancies such as melanoma and multiple myeloma.

Apoptosis in Proliferating, Senescent, and Immortalized Keratinocytes

Skin provides an attractive organ system for exploring coordinated regulation of keratinocyte (KC) proliferation, differentiation, senescence, and apoptosis. Our main objective was to determine whether various types of cell cycle arrest confer resistance to apoptosis. We postulated that KC cell cycle and cell death programs are tightly regulated to ensure epidermal homeostasis. In this report, simultaneous expression of cyclin-dependent kinase inhibitors (p15, p16, p21, and p27), a marker of early differentiation (keratin 1), mediators of apoptosis (caspases 3 and 8), and NF-κB were analyzed in three types of KCs. By comparing the response of proliferating, senescent, and immortalized KCs (HaCaT cells) to antiproliferative agents followed by UV exposure, we observed: 1) Normal KCs follow different pathways to abrupt cell cycle arrest; 2) KCs undergoing spontaneous replicative senescence or confluency predominantly express p16; 3) Abruptly induced growth arrest, confluency, and senescence pathways are associated with resistance to apoptosis; 4) The death-defying phenotype of KCs does not require early differentiation; 5) NF-κB is one regulator of resistance to apoptosis; and 6) HaCaT cells have undetectable p16 protein (hypermethylation of the promoter), dysfunctional NF-κB, and diminished capacity to respond to antiproliferative treatments, and they remain highly sensitive to apoptosis with cleavage of caspases 3 and 8. These data indicate that KCs (but not HaCaT cells) undergoing abruptly induced cell cycle arrest or senescence become resistant to apoptosis requiring properly regulated activation of NF-κB but not early differentiation.

Superimposed histologic and genetic mapping of chromosome 9 in progression of human urinary bladder neoplasia: implications for a genetic model of multistep urothelial carcinogenesis and early detection of urinary bladder cancer

The evolution of alterations on chromosome 9, including the putative tumor suppressor genes mapped to the 9p21-22 region (the MTS genes), was studied in relation to the progression of human urinary bladder neoplasia by using whole organ superimposed histologic and genetic mapping in cystectomy specimens and was verified in urinary bladder tumors of various pathogenetic subsets with long-term follow-up. The applicability of chromosome 9 allelic losses as non-invasive markers of urothelial neoplasia was tested on voided urine and/or bladder washings of patients with urinary bladder cancer. Although sequential multiple hits in the MTS locus were documented in the development of intraurothelial precursor lesions, the MTS genes do not seem to represent a major target for p21-23 deletions in bladder cancer. Two additional tumor suppressor genes involved in bladder neoplasia located distally and proximally to the MTS locus within p22-23 and p11-13 regions respectively were identified. Several distinct putative tumor suppressor gene loci within the q12-13, q21-22, and q34 regions were identified on the q arm. In particular, the pericentromeric q12-13 area may contain the critical tumor suppressor gene or genes for the development of early urothelial neoplasia. Allelic losses of chromosome 9 were associated with expansion of the abnormal urothelial clone which frequently involved large areas of urinary bladder mucosa. These losses could be found in a high proportion of urothelial tumors and in voided urine or bladder washing samples of nearly all patients with urinary bladder carcinoma.

Role of NF-κB in the Apoptotic-resistant Phenotype of Keratinocytes

Several studies point to a role for NF-κB in modulating epidermal thickness and apoptotic susceptibility of keratinocytes. When phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) are topically applied, prominent epidermal thickening occurs, and exposure to interferon (IFN)-γ promotes increased epidermal thickness producing psoriatic lesions. While keratinocytes derived from psoriatic plaque resist apoptosis, and combination of TPA and IFN-γ activates NF-κB, the molecular mechanism linking NF-κB activation and keratinocyte apoptosis resistance was unknown. Therefore, we examined the ability of IFN-γ plus TPA to influence NF-κB activity, gene expression, and response to UV light-induced apoptosis. These responses in normal keratinocytes were compared with immortalized keratinocytes (HaCaT cells). Exposure of normal keratinocytes to IFN-γ plus TPA produced a synergistic activation of NF-κB, compared with when each reagent was used individually. Normal keratinocytes when exposed to IFN-γ plus TPA acquired a resistance to UV light-induced apoptosis, which was dependent on NF-κB because expression of a dominant negative form of IκBα overcame the resistance. Compared with normal keratinocytes, HaCaT cells have a dysfunctional constitutive NF-κB signaling pathway not induced by IFN-γ and TPA, rendering HaCaT cells highly susceptible to UV-induced apoptosis. Thus, immortalized HaCaT cells have an abnormal constitutive and dysfunctional NF-κB signaling system. These results provide evidence that activation and proper regulation of NF-κB is essential for acquisition of an apoptotic-resistant phenotype for epidermal-derived keratinocytes.

Avoiding premature apoptosis of normal epidermal cells

DAVID LAWRENCE, ZAHRA SHAHROKH, SCOT MARSTERS, KIRSTEN ACHILLES, DANNY SHIH, BARBARA MOUNHO, KENNETH HILLAN, KLARA TOTPAL, LAURA DEFORGE, PETER SCHOW, JEFFREY HOOLEY, STEVE SHERWOOD, ROGER PAI, SUSAN LEUNG, LOLO KHAN, BRIAN GLINIAK, JEANINE BUSSIERE, CRAIG A. SMITH, STEPHEN S. STROM, SEAN KELLEY, JUDITH A. FOX, DEBORAH THOMAS & AVI ASHKENAZI Genentech Incorporated, South San Francisco, California, USA Immunex Corporation, Seattle, Washington, USA University of Pittsburgh, Pittsburgh, Pennsylvania, USA Email: aa@gene.com 1. Wiley, S.R. et al. Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 3, 673–682 (1995). 2. Pitti, R.M. et al. Induction of apoptosis by Apo-2 Ligand, a new member of the tumor necrosis factor receptor family. J. Biol. Chem. 271, 12697–12690 (1996). 3. Ashkenazi, A. et al. Safety and anti-tumor activity of recombinant soluble Apo2 ligand. J. Clin. Invest. 104, 155–162 (1999). 4. Walczak, H. et al. Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo. Nature Med. 5, 157–163 (1999). 5. Gliniak, B. & Le, T. Tumor necrosis factor-related apoptosis-inducing ligand’s antitmor activity in vivo is enhanced by the chemotherapeutic agent CPT-11. Cancer Res. 59, 6153–6158 (1999). 6. Roth, W. et al. Locoregional Apo2L/TRAIL eradicates intracranial human malignant glioma xenografts in athymic mice in the absence of neurotoxicity. Biochem. Biophys. Res. Commun. 265, 1999 (1999). 7. Chinnaiyan, A.M. et al. Combined effect of tumor necrosis factor-related apoptosis-inducing ligand and ionizing radiation in breast cancer therapy. Proc. Natl. Acad. Sci. USA 97, 1754–1759 (2000). 8. Jo, M. et al. Apoptosis induced in normal human hepatocytes by tumor necrosis factor-related apoptosis-inducing ligand. Nature Med. 6, 564–567 (2000). 9. Nagata, S. Steering anti-cancer drugs away from the TRAIL. Nature Med. 6, 502–503 (2000). 10. Hymowitz, S.G. et al. Triggering cell death: the crystal structure of Apo2L/TRAIL in a complex with death receptor 5. Molec. Cell 4, 563–571 (1999). 11. Hymowitz, S.G. et al. A unique zinc-binding site revealed by a high-resolution X-ray structure of homotrimeric Apo2L/TRAIL. Biochemistry 39, 633–640 (2000). 12. Bodmer, J.-L., Meier, P., Tschopp, J. & Schneider, P. Cysteine 230 is essential for the structure and activity of the cytotoxic ligand TRAIL. J. Biol. Chem. 275, 20632–20637 (2000). 13. Schneider, P. et al. Conversion of membranebound Fas(CD95) ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of liver toxicity. J. Exp. Med. 187, 1205–1213 (1998). 14. Nicoletti, I., Migliorati, G., Pagliacci, M.C., Grignani, F. & Riccardi, C. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J. Immunol. Methods 139, 271–279 (1991). 15. Kischkel, F.C. et al. Apo2L/TRAIL-dependent recruitment of endogenous FADD and caspase-8 to death receptors 4 and 5. Immunity 12, 611–620 (2000). 16. Ashkenazi, A. & Dixit, V.M. Apoptosis control by death and decoy receptors. Curr. Opin. Cell Biol. 11, 255–260 (1999).

Regulation of apoptosis by p53 in UV-irradiated human epidermis, psoriatic plaques and senescent keratinocytes.

The carcinogenic effects of sunlight in human epidermis may be thwarted by either: transient growth arrest and repair of DNA photodamage in keratinocytes (KCs); elimination of KCs with damaged DNA via apoptosis; or by stimulating a senescence switch whereby KCs become irreversibly growth arrested. Using normal human skin organ cultures and living epidermal equivalents, we demonstrate that in the proliferative basal layer, removal of KCs via apoptosis had a rapid onset (beginning within 2 h) following UV-light exposure generating progressively greater numbers of KCs with thymine dimers as the dose of UV-light was increased; involved induction of Apaf-1, activation of caspase-3, and was dependent on p53 activation as addition of a p53 chemical inhibitor blocked the apoptotic response. Suprabasal layer KCs underwent apoptosis at much later time points (>8 h). KCs in the basal layer repaired DNA damage more rapidly than KCs in suprabasal layers. Steady state levels of p53 increased in irradiated cells, and the increase was accompanied by phosphorylation of serine 9 and serine 15, but not serine 6 residues. By contrast, cultured KCs undergoing spontaneous replicative senescence were resistant to UV-induced apoptosis. Senescent KCs constitutively contained low levels of p53, which were neither increased nor phosphorylated or acetylated after UV-exposure and possessed minimal DNA binding activity, indicative of functional inactivation. Furthermore, treatment of senescent KCs with DNA damaging agent adriamycin did not result in activation of latent p53 or apoptosis. When KCs within psoriatic plaques were examined, they resembled senescent KCs in that they expressed p53, which was not phosphorylated or acetylated. Thus, UV-light induces DNA damage in human epidermal KCs triggering p53 activation, and subsequent apoptosis involving distinct cell layers and kinetics. However, the lack of p53 activation as seen in senescent KCs and psoriatic plaques, is associated with a relative resistance of KCs to UV-induced apoptosis. In conclusion, the sensitivity and resistance of KCs to apoptosis depends not only on the location within various layers of epidermis and levels of p53, but may also involve p53 activation via post-translational modifications.

Genetic modeling of human urinary bladder carcinogenesis

We developed a model of human urinary bladder cancer progression from in situ precursor lesions to invasive carcinoma using whole organ histologic and genetic mapping. The model represents a high‐density and detailed analysis regarding allelic losses on chromosomes 4, 8, 9, 11, and 17 as revealed by testing of 234 samples obtained from five cystectomy specimens. The samples corresponded to microscopically identified intraurothelial precursor conditions ranging from dysplasia to carcinoma in situ and invasive cancer. The initial analysis of paired normal and tumor DNA samples disclosed allelic losses in 72 of 225 tested hypervariable DNA markers. Subsequent use of these markers on all mucosal samples revealed that 47 had alterations with a statistically significant relation to urothelial neoplasia. The allelic losses clustered in 33 distinct chromosomal regions, indicating the location of putative tumor suppressor genes involved in the development and progression of urinary bladder cancer. Some of the markers with statistically significant allelic losses mapped to the regions containing well‐characterized tumor suppressor genes but many were located in previously unknown loci. The majority of statistically significant allelic losses (70%) occurred early in low‐grade intraurothelial dysplasia, and some of them involved adjacent areas of morphologically normal mucosa preceding the development of microscopically recognizable precursor lesions. The remaining 30% of markers developed allelic losses in the later phases of urothelial neoplasia, implicating their involvement in progression to invasive disease. Markers exhibiting allelic losses in early phases of urothelial neoplasia could be used for detection of occult preclinical or even premicroscopic phases of urinary bladder cancer, whereas markers that showed allelic losses in the later phases of the process could serve as indicators of progression to invasive disease. The approach used in this study facilitates genome‐wide modeling of cancer progression and provides important chromosomal landmarks for more specific studies of multistep urinary bladder carcinogenesis. Genes Chromosomes Cancer 27:392–402, 2000.

The proapoptotic tumor suppressor protein kinase C- δ is lost in human squamous cell carcinomas

Protein kinase C (PKC)-δ is proapoptotic in human keratinocytes, and is downregulated or inactivated in keratinocytes expressing the activated Ha-ras oncogene, making it a candidate tumor suppressor gene for squamous cell carcinoma (SCC). We evaluated the significance of PKC-δ loss in transformed human keratinocytes using tumorigenic HaCaT Ras II-4 cells that have significantly reduced PKC-δ levels. Re-expression of PKC-δ by retrovirus transduction caused an increase in apoptosis and growth inhibition in culture. The growth inhibition induced by PKC-δ could be partially reversed by Bcl-xL expression, indicating that apoptosis was in part responsible for PKC-δ-induced growth inhibition. PKC-δ re-expression suppressed the tumorigenicity of HaCaT Ras II-4 cells in nude mice (P<0.05), and the small tumors that did form contained elevated levels of activated caspase-3, indicating increased apoptosis. In addition, we found that 29% (12/42) of human Bowens disease (squamous carcinoma in situ) or SCC cases had absent or reduced PKC-δ when compared to the surrounding normal epidermis. These results indicate that PKC-δ inhibits transformed keratinocyte growth by inducing apoptosis, and that PKC-δ may function as a tumor suppressor in human SCCs where its loss in cells harboring activated ras could provide a growth advantage by conferring resistance to apoptosis.

Superimposed histologic and genetic mapping of chromosome 17 alterations in human urinary bladder neoplasia

Multistep alterations of chromosome 17 in the progression of human urinary bladder neoplasia were studied by superimposed histologic and genetic mapping. The p53 gene was included in the analysis as a model tumor suppressor gene that is frequently involved in urothelial carcinogenesis. The strategy provided a systematic approach to the study of multistep genomic alterations that occur as neoplasia progresses from precursor intraurothelial conditions to invasive cancer. This was accomplished by sampling the entire mucosa of the organ and displaying microscopically identified invasive cancer and precursor conditions in the form of a histologic map. Subsequent isolation of DNA provided a set of samples in which the search for genetic alterations was performed and superimposed on the histologic map. This approach disclosed multifocal allelic losses of chromosome 17 in the early preinvasive phases of urothelial neoplasia. The alterations were predominantly confined to the p12 – 13, q22 – 11 and q24 – 25 regions. Mutations and allelic losses of the p53 gene were mapped to early preinvasive phases of urothelial neoplasia. The data provide detailed analysis of chromosome 17 allelic losses that occur in the development and progression of urothelial neoplasia and represent the first step for genome-wide modeling of multistep human urothelial carcinogenesis.

Knockdown of p53 levels in human keratinocytes accelerates Mcl-1 and Bcl-xL reduction thereby enhancing UV-light induced apoptosis

Ultraviolet (UV) light exposure is a common cause of epithelial-derived skin cancers, and the epidermal response to UV-light has been extensively studied using both mouse models and cultured human keratinocytes (KCs). Elimination of cells with UV-induced DNA damage via apoptosis provides a powerful mechanism to minimize retention or expansion of genetically abnormal cells. This cell editing function has largely been ascribed to the biological role of the p53 tumor suppressor gene, as mutations or deletions involving p53 have been linked to skin cancer development. Rather than introducing mutations, or using cells with complete loss of wild-type p53, we used an siRNA-based approach to knockdown, but not eliminate, p53 levels in primary cultures of human KCs followed by UV-irradiation. Surprisingly, when p53 levels were reduced by 50–80% the apoptosis induced by exposure to UV-light was accelerated and markedly enhanced (two- to three- fold) compared to control siRNA treated KCs. The p53 siRNA treated KCs were characterized by elevated E2F-1 levels accompanied by accelerated elimination of the Mcl-1 and Bcl-xL antiapoptotic proteins, as well as enhanced Bax oligomerization. Forced overexpression of either Mcl-1 or Bcl-xL reduced the UV-light enhanced apoptotic response in p53 siRNA treated KCs. We conclude that p53 not only can provide proapoptotic signals but also regulates a survival pathway influencing Mcl-1 and Bcl-xL levels. This overlooked survival function of p53 may explain previous paradoxical responses noted by investigators using p53 heterozygous and knockout mouse models, and opens up the possibility that not all liaisons within the cell involving p53 necessarily represent fatal attractions.