Indo-French symposium on apoptosis and multidrug resistance

Leukemia Research Vol. 21, No. 9, pp. 899-902, 1997. 0 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain 014552126/97 $17.00 + 0.00

Pergamon PII: SO1452126(97)00066-O

CONCISE REPORT INDO-FRENCH SYMPOSIUM ON APOPTOSIS AND MULTIDRUG RESISTANCE M. Bhargava* and Amu Therwatht *Professor and Head, Department of Hematology, AIIMS, New Delhi 110029, India and TProfessor, Laboratoire d’oncologie Moleculaire, UniversitC Paris VII, Paris, France (Received 25 March 1997.Accepted 23 April 1997)

these subjects for a clearer picture to emerge. M. F. Poupon presented data on the defects of hexokinase metabolism occurring as a consequence of loss of chromosome 10 in gliomas, and the sensitivity of intracellular distribution of hexokinase to variations of pH which can be effectively used for tumor kill by pharmacological acidification of the tumor cells. Methionine metabolism deficiency in some gliomas can also be used to induce a growth inhibition. S. Sinha discussed LOH of 17~ 13.3 locus occurring as an independent event in a majority of higher grades of astrocytic tumors and its probable involvement in the transition from low grade to higher grades. LOH of 17p 13.1 locus occurred in the majority of astrocytic tumors irrespective of the grade. B. R. Das’s data regarding the regulation of the ~53 gene in carcinoma breast showed no correlation between the ~53 protein accumulation and the mutations in exon 5-8. Thirty-three percent of female patients had mutations, localized either in exon 5 or 6. In males, mutation was detected in 90% of cases, localized exclusively to exon 6. Preliminary data presented on four different nuclear proteins that bind to the ~53 suggested that a differential binding of these nuclear proteins to p.53 promoter in normal and tumor tissue might be responsible for a transcriptional deregulation of the ~53 gene. S. Chevillard highlighted the relevance of measuring the S phase fraction and MDR phenotype as a potential early predictive marker of tumor resistance to neoadjuvant chemotherapy (with or without Adriamycin) in breast cancer. While the S phase response was found to be associated with clinical response in both regimes, MDR expression was predictive only in patients on the FAC (5-FU, Adriamycin Cyclophosphamide) regime. Tumors negative for MDR expression on both day 0 and day 8 of therapy had a better prognosis. K. N. Naresh spoke on the relevance of apoptosis index in squamous cell carcinoma of the tongue (stage Tl, NO) as a predictor of metastatic phenotype and

Symposium held at IUCAA, University of Pune, Pune, India, 5-l 1 February 1997 The state of the art knowledge in the rapidly expanding fields of apoptosis and multidrug resistance (MDR) and their importance in anticancer drug development, was the subject of the meeting held in Pune, India, from 5 to 1997. The meeting was organized by 11 February Professor Manorama Bhargava from India and Professor Amu Therwath from Paris on behalf of the Indo-French Centre for Promotion of Advanced Research. The aim of the meeting was to bring together the foremost scientists working on basic and clinical aspects of apoptosis and MDR for an exchange of ideas and to stimulate further development and collaboration in these exciting areas between Indian and the French scientists. The various sessions covered topics such as oncogenes and apoptosis in cancer, apoptosis in development and immunity, modulators of apoptosis, MDR and its circumvention, and drug design. The opening presentation of the symposium by Dr S. S. Aggarwal (SGPGIMS, Lucknow, India) set the scene by giving an introduction and a broad overview on apoptosis, the nature of signal cascades and genes involved, and its role in health and disease. The first session of the meeting was on Oncogenes and Cancer (Chairperson: Amu Therwath). Molecular genetics of acute lymphoblastic leukemia (ALL) was outlined by M. Bhargava, highlighting the significant differences in the biology of ALL in India as compared to the West in terms of immunophenotype (a high incidence of T-ALL) and genotype (differences in immunoglobulin and T cell antigen receptor (TCR) genes, ALL1 and BCR-ABL gene rearrangements). Commenting on the poor survival of these patients she pointed out that it was necessary/imperative to study drug resistance and lesions in the apoptotic pathway in Correspondence to: M. Bhargava, Professor and Head, Departmentof Hematology, AIIMS, New Delhi 110029,India. 899

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tumor progression at presentation. The ‘turnover index’ seeksto combine apoptotic index and proliferation index in tumors. For small (3 mm) thickness early tumors, when the apoptotic index was ~3% the survival was lOO%, while when the index was >3% survival was < 40%. The index is relevant only for smaller tumors. Apoptosis index emergedas an independentpredictor of lymph node metastasis. N. Singh emphasized the need to use as many parameters as possible to demonstrate/identify the phenomenon of apoptosis, although morphological characteristicswere still the most widely and uniformly accepted ones. Her results showed that tumor necrosis factor a (TNFu) induced apoptosis in mouse epidermal JB6 RTlOl cells using TNFa-sensitive and -resistantRT 101 cells and that the apoptosisappearedto be mediated by C-Jun/AP-1. She also presented her findings demonstrating the involvement of C-Myc, C-Jun, antioxidant enzymes and poly ADP ribosylation in Adriamycin-induced apoptosis. M. Katdare described a system of immortalized nontumorigenic human mammary 184-B5 cells and HER-Y neu protooncogene initiated 184-B5/HER cells. The transfectedcells showed aberrant growth properties and a differential responseto three phytochemicals-indole 3-carbinol (13C), (-)-epigallocatechin (EGCG) and genistein-obtained from cruciferous vegetables,black and green tea, and soya respectively. Non-toxic dosesof the three phytochemicals were able to induce apoptosis in transfectedcells (although the doseswere higher than for non-transfected cells). Apoptosis induction correlated with the decreaseof Bcl-2 and PCNA (Proliferating cell nuclear antigen) proteins. The second session of the meeting was devoted to Apoptosis and Cancer (Chairperson: P. Sax-thou).A. Khar spoke on apoptosisin tumor regression,emphasizing the role of natural killer (NK) cells in induction of apoptosis in AK-5 cells. In in vivo experiments in rats, NK cells from naive animals could induce apoptosis of AK-5 cells and the activity could be augmented by treatment with interleukin 2 @-2)/B-12. Cysteine proteaseswere implicated in the execution of apoptosis. The NK cell activity could be reversedby transfection of AK-5 cells with Ned-2 gene in antisenseorientation and with the bCl-2 gene. M. R. Pillai spoke about the importance of apoptotic index and expression of P53, Bcl-2 and P-glycoprotein in predicting response to therapy in cases of pediatric ALL. A low rate of spontaneousapoptosis,overexpressionof P53, Bcl-2 or P-glycoprotein at presentation implied a poor response. S. G. A. Rao highlighted novel strategiesto modulate apoptosis in the apoptosis-resistantblasts seen during the blastic (terminal) phase of chronic myeloid leukemia, Inhibiting the ~210 and ~145 kinases activity by genistein and quercitin can result in the exit of the cells

out of the cell cycle. Genistein treatment affected only the tyrosine kinase activity, whereas quercetin had inhibitory action on protein kinase C (PKC) also. Apoptosis predominated in quercetin treatment whereas genistein resulted in differentiation as well. B. C. Das spoke on the role of human papillomavirus (HPV) and apoptosis in cervical cancer and proposed strategiesthat could lead to an understandingof the role played by E6 and E7 proteins of HPV in the regulation of cell proliferation and apoptosis: their respective interaction with ~53 and rb-1 genes. In the session on Apoptosis and Development (Chairperson: S. G. A. Rao) D. Bhartiya presented evidence to show that apoptosishas a functional role in the endometrial cycle using ovariectomized mice and sequential administration of estrogen and progesterone. A critical issue that was raised in the discussion of the presentation was that caution is needed in the interpretation of electron micrographs.R. R. Dighe presented data on induction of apoptosis in the germ cells by blocking the action of two key hormones, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). By purifying the different cell populations, administration of LH antiserum could induce apoptosis in pachytene spermatocytesand round spermatid-enriched cell populations. Inhibition of FSH action also resulted in induction of apoptosis in pachytene spermatocytes. The role of transforming growth factor p (TGFB) as a modulator of apoptosis,and its relation to the action of testosteronein the male accessoryglands, was discussed by P. Kondaiah. K. Subba Rao explained how typical apoptotic DNA fragmentation induced by glutamate or h4NNG (Nmethyl-N’nitro-N-nitrosoguanidine) could not be seen in a 2-year-old neurone despite harboring at least 600 double-stranded breaks. He therefore proposed that apoptosis may not be the cause of age-relatedneuronal degeneration. S. ManduE explained the role of three apoptosisrelated genes, rpr, hid and grim in Drosophila, and presented his findings demonstrating that benzamide feeding or rearing at low temperatures restored the number of ommatidia in Bar mutant flies possibly because benzamide and low temperature can prevent apoptosis of ommatidia. P. D. Gupta elaborated the role of apoptosis during embryonic development, cell turnover in the intestine, and tumor cell depletion and highlighted the various parametersincluding morphology, activation of DNase, activation of transglutaminaseand DNA breakdown in the assessmentof apoptosis. The opening presentation of the sessionon Modulators of Apoptosis (Chairpersons: J. Robert and N. Singh) by E. Salary discussedthe role of caspasesand Fas receptor in cytotoxic drug-induced tumor cell death.

Indo-french symposium on apoptosis and multidrug resistance

He investigated etoposide and Fas-induced apoptosis in human leukemic cell line U937. A cleavage of procaspase 3 (CPP32) was observed while another procaspase,ICH-IL, was unchanged.The study of CASP geneexpressionin U937 cells treated with etoposideand the modulation of this expression by Bcl-2 and tetrapeptide proteaseinhibitors indicated that (1) CASP gene expression is regulated by factors sensitive to activated caspases,and (2) post-transcriptional mechanisms are essential in the regulation of procaspase expression. S. Metkur’s presentation focused on FasFasL interactions and their involvement in the induction of apoptosisin Hodgkin’s disease(HD). Three cell lines, L428, KMH2 and HDLM2, derived from pleural effusion of HD patients were found to strongly express Fas but showed no expression of Fas-L. G. Laurent presentedinteresting results on how the SPM (sphingomyelin)-ceramide pathway can be activated by daunorubicin (DNR) to induce apoptosis in U937 leukemic cells, and that antitumor agents like DNR and ionizing radiation cannot generateapoptosisin resistant leukemic cells due to the absenceof the SPM-ceramidepathway. The ceramide signaling pathway may be altered depending on Bcl-2 expression, antioxidative status and PKC activity. The blockage of ceramide-mediated apoptotic pathway may play a role in the developmentof resistance in leukemic cells. Evidence was also offered to suggest that SPMase activation may occur independently of DNA lesions. P. Surthou elaboratedthe complex signaling cascades triggered by multiple receptors in the samecell, during the activation of a B lymphocyte. S. E. Husnain highlighted the therapeutic potential of ~35 gene product as an effective protector against ROS (Reactive Oxygen Species)-inducedapoptosis. G. Punde elaborated upon the use of flowcytometer for various markers of apoptosis using either single or double parameter analysis. B. S. Shunkur showed data where post-irradiation serum deprivation protects cells from apoptosis.L. Srinivus emphasizedthe role of lipid peroxides as antiapoptotic agents/tumorpromoters. In the session on Apoptosis and Immunity (Chairperson: M. Bhargava) P. Surthou dissected out the pathways involved in apoptosis in B lymphocytes using the WEHIB lymphoma model. The role of protein tyrosine kinase substrate P7SHS’was discussed in the transmission of positive and negative signals from surface immunoglobulins to the nucleus. Transfection of HSI gene in mutant WEHIcells led to the recovery of full sensitivity. NF-rcB and c-myc appeared to be some of the main genes involved in regulating apoptosis in B cells. Using y/S T cells from the peripheral blood and tumor tissue of esophagealcancer patients, S. V. Chiplunkar showed that ylS T cells act as killer cells and lyse oral tumor cells via recognition of

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heat shock protein (hsp 60) ligand on the tumor cell surface. She further demonstratedthat growth of y/6 T cells becomesself-limiting in both the compartmentsby the antigen-induced apoptosis. The study demonstrated that apoptosismay be induced via hsp 60, and Fasligand may not be involved. The penultimate session was on Multidrug Resistance and Drug Design (Chairpersons:J. P. Marie, U. N. Das, K. N. Thimmaiah and E. Solary). J. C. Jurdillier discussed the hypersecretory process of lysosomal enzymes by MDR cells (CEM/VLBi& in comparison with drug-sensitive human lymphoblastic leukemic CEM cells. CEM/VLBloo cells showed increased secretionand lower content of iV-acetyl glucosaminidase (NAGA) and fi galactosidasethan CEM cells and its modulation by verapamil. NIH 3T3 cells transfected with full-length MDR-1 cDNA showedsimilar results to CEMNLB ioo cells, while the mutated MDR-1 cDNA transfected NIH 3T3 cells did not demonstrate any alteration in lysosome activity. The role of P-gp in metastasisand its correlation with proteolysis by MDR (+) cells was discussed.Based on his experience of the French multicentric trial J. P. Marie made the following recommendationsto be kept in mind while measuring MDRl proteins: (1) for measurementof level of P-gp expression by fluorescence-activatedcell sorter (FACS) analysis the ratio of fluorescence intensity with monoclonal antibody to IgG control antibody should be used; (2) in immunocytochemical analysis of P-gp at least two antibodies should be used. JC-1 probe can be used to increasethe sensitivity of tests for P-gp; and (3) calceinAM is useful for MRP detection. IV. F. Poupon talked about genomic instability and the effect of ~53 mutations on amplification of genes involved in metastasis and in resistance to antimetabolites in the human LoVo colon carcinoma cell line. Two mutations in ~53, 175his and 273his were associated with metastatic potential. She tested the resistance to antimetabolite PALA (N-phosphonoacetyl-L-aspartate) due to an amplification of the CAD gene in ~53 wild type or mut p53-transfected LoVo cells and showed a higher frequency of resistance in the mut p534ransfected cells. These mut p534ransfected cells showed a high metastatic potential when grafted orthotopically in the colon of nude mice. J. Robert discussed the identification of DNA sequences which could be specifically targeted by topoisomerase II-interfering drugs based on the hypothesis that there exist genomic DNA sequenceswhere the formation of drug-induced cleavable complexes occurs preferentially. Such sequencesmay be formed at the level of genesinvolved in cell proliferation and thus explain drug specificity and efficiency. J. P. Marie compared the clinical significance of MDR phenotype to other clinical and biological

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prognostic factors in newly diagnosed acute myelogenous leukemia (AML) patients. MDRl overexpression emerged as an important prognostic factor in both uniand multivariate analyses. The significance of MRP (multidrug resistanceassociatedprotein) and LRPMVP (Lung resistanceprotein/major vault protein) in AML is still under investigation. S. Gurbuxuni in his talk on the expressionof genesimplicated in MDR in ALL said that although the role of P-gp in AML is well documentedit is not so in the case of ALL. His study showed no correlation between MDRl and MRP values with treatment outcome.It was suggestedthat this is possibly because the cortico-steroids that play a major role in treatment of ALL are not substratesfor either MDRl or MRP. Sequential assessmentof MDR phenotype and measurementof S phase fraction as predictive markers of breast cancer responseto neoadjuvant chemotherapy was presentedby S. Chevillard. The surprising finding was that caseswith overexpressionof MDRl at the time of presentation had a better prognosis than those with lower but progressively increasing MDRl expression.It was also noted that ~53 expression has no value in predicting the outcome of treatment in breast cancer. R. Ralhan presented evidence for the correlation of overexpression of P-gp with tumor progression in oral cancer. A significant association of P-gp and ~53 coexpression was shown to correlate directly with chemoresistance, aggressive phenotype of the tumor cells and tumor progression. The GST-rcgene, which is also implicated in the phenomenon of drug resistance, was shown to be overexpressed,more so in recurrent rather than primary tumors. Mutant ~53 was shown to activate the promoter of the MDRl gene. A differential expression of ~53 was observed during the process of oral tumorigenesis. J. C. Jurdillier, talking about decreasedinflux as the primary cause of MDR, showed that liposomes formed from phosphatidylinositol (PI) and cardiolipin increased the influx of vincristine in resistant cells but did not affect the efflux of the drugs. There was no cytotoxic effect due to liposomes alone. It is possible that PI and cardiolipin alter the membranefluidity and then enhance any uptake. In an attempt to develop a new strategy for circumventing MDR, A. Juyakrishnan shared his experience regarding the drug-encapsulated microspheresprepared using the milk protein casein and the naturally occurring polysaccharidechitosan which were loaded with mitoxantrone. Water-soluble analogs of campothecins containing a lactone ring were examined as the putative inhibitors for topoisomeraseI by J. Robert. It was observed that the predominant form of SN-38 circulating in plasma was

the lactone formed as a result of the enzymic conversion by carboxyl esterase,which also explains the clinical activity of irinotecan. The other two metabolites of irinotecan, however, did not show any significant activity on topoisomerase I-DNA complexes. A. S. Juvekur hypothesized that a new drug combination consisting of mitomycin and cisplastin can be very effective in the treatment of carcinoma of the larynx, particularly in cases with increased MDR marker positivity (overexpressionof P-gp, PKC and GST-n). The last sessionof the meeting was on Circumvention of MDR (Chairperson: J. C. Jardillier). K. N. Thimmaiah presentedhis work on reversal of multidrug resistanceby phenoxazines, as compared to verapamil. Using drug-resistant and -sensitive cell lines the effect of phenoxazine on accumulation of vinblastine was demonstrated. Lipophilicity increased vinblastine uptake. In colon adenocarcinoma cell lines morpholine derivatives of phenoxazines showed greater accumulation of the drug. Results showed that these modulators work independently of P-glycoprotein but could involve an alteration in drug influx and were devoid of any significant cytotoxicity. Cr.N, Das presentedhis work on the cytotoxicity of essentialfatty acids on tumor cells in vitro. y-Linolenic acid (GLA) and eicosapentanoicacid (EPA) potentiated the cytotoxic action of anticancer drugs. The mechanism of action was through enhanced lipid peroxidation and superoxide generation. Injection of GLA in patients with recurrent gliomas and hepatomasdemonstratedregression of these tumors. E. So&r-y presentedthe results of a phase III multicentric clinical study to evaluate whether the responserate of poor-risk acute leukemic patients to conventional chemotherapy (mitoxantrone and cytarabine) could be improved by quinine. A complete response was observed in 52.8% of patients from the quinine-treated group versus 45.5% in the control group. Encouraging results were observed in refractory AMLs and blastic transformation of myelodysplastic and myeloproliferative syndrome.The complete responserate was higher in P-glycoprotein-positive cases.The study demonstrated that ability of serumto increasemitoxantrone accumulation in an MDR-positive leukemic cell line was linearly related to quinine level in the serum of quinine-treated patients. The study identified quinine as a clinically relevant MDR-reversing agent. S. Padhye discussedhis experience of screening Indian folk medicine for reversal of drug resistance. He further added that ‘Chits& (Plumbago zeylanica) has been mentioned as a remedy for diseasesrequiring long-term therapeutic treatment, especially when these disorders becamenonresponsive and refractory to the administered antibiotics.