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Oncology Research, Volume 14, pp. 321-324
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Copyright © 2004 Cognizant Comm. Corp.
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Diagnostic, Endocrinological, Behavioral, and DNA Ploidy Differences Between Squamous Cell and Adenomatous Carcinoma of the Cervix Uteri

Annika Kristina Lindström,1,2 Dan Hellberg,3,4 Torbjörn Bäckström,5 and Ulf Stendah1,2

1The Ventrum Clinic, Bjursås, Sweden
2Department of Gynecological Oncology, Norrlands University Hospital, Umeå, Sweden
3Department of Obstetrics and Gynecology, Falun Hospital, Falun, Sweden
4Centre for Clinical Research, Falun, Sweden
5Department of Obstetrics and Gynecology, Norrlands University Hospital, Umeå, Sweden

The objective of this study was to compare behavioral and hormonal risk factors, and cancer characteristics in squamous cell and adenocarcinoma of the cervix uteri. The study included 45 women with adenocarcinoma and 190 consecutive women with squamous cell carcinoma of the cervix uteri diagnosed between 1984 and 1990. In addition to routine diagnostic procedures, DNA from biopsies was analyzed with flow cytometry to identify the S-phase fraction as a measure of cancer growth. Serum progesterone and estradiol were measured, and ever hormonal use and smoking were recorded. Ten-year mortality was estimated. There was no significant difference in age at diagnosis. Women with adenocarcinoma, compared with those with squamous epithelial carcinoma, had a significantly shorter duration of ever contraceptive use, were less likely to smoke (nonsignificant), had a lower cancer stage, were less likely to have an S-phase fraction of more than 16%, and had a higher progesterone value (if premenopausal). The results suggest that there are some different risk factors in adenocarcinoma and squamous cell carcinoma of the cervix uteri.

Key words: Cervix neoplasms; Adenocarcinoma; Squamous cell carcinoma

Address correspondence to Dr. Annika Kristina Lindström, Skovägen 2, S-79021 Bjursås, Sweden. Tel: +46 23 40310; E-mail: ventrum@telia.com

Oncology Research, Volume 14, pp. 325-330
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Copyright © 2004 Cognizant Comm. Corp.
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Effect of Association of Temozolomide With Other Chemotherapic Agents on Cell Growth Inhibition in Glioma Cell Lines

Marco Balzarotti, Emilio Ciusani, Chiara Calatozzolo, Danilo Croci, Amerigo Boiardi, and Andrea Salmaggi

Laboratory of Clinical Investigation, Department of Neurology, National Neurological Institute "C. Besta." Via Celoria 11, 20133 Milan, Italy

Despite progresses in surgery and treatments of malignant gliomas, prognosis of these tumors remains poor, with a median life expectancy of 12 months in glioblastomas. Chemotherapy (mostly with nitrosoureas) has been demonstrated to prolong overall survival, but the entity of this improvement is slight and disease recurrence/progression is the rule, stressing the need for multimodality treatment. In this work we investigated the effect of association of temozolomide (TMZ), an orally bioavailable alkylating agent, with three chemotherapeutic drugs, liposomal doxorubicin (DOXO), cis-platinum (CDDP), and topotecan (TP), on cell growth of A172, U373, U138, U87, and SW1783 (all human glioma cell lines). Results indicate a synergistic effect (CI < 1) of TMZ in association with liposomal DOXO and CDDP on cell growth inhibition in most of the studied cell lines (A172, U373, U138, U87). Synergistic effect also has been obtained after treatment of A172 and U373 with TMZ and TP in association. In conclusion, our results confirm the potential effect of association of chemotherapic drugs with different mechanisms of action in the treatment of gliomas.

Key words: Temozolomide; Doxorubicin; Cis-platinum; Topotecan; Glioma; Locoregional

Address correspondence to Andrea Salmaggi, M.D., Department of Neurology, National Neurological Institute "C. Besta," Via Celoria, 11, 20133 Milan, Italy. Tel: +39 02 2394247; Fax: +39 02 2394535; E-mail: salmaggi@istituto-besta.it

Oncology Research, Volume 14, pp. 331-343
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Copyright © 2004 Cognizant Comm. Corp.
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Age-Related Differences in Vincristine Toxicity and Biodistribution in Wild-Type and Transporter-Deficient Mice

Toshinari Muramatsu,1* Dennis R. Johnson,1* Rick A. Finch,1 Linda K. Johnson,3 Janine J. Leffert,2 Z. Ping Lin,1 Giuseppe Pizzorno,1,2 and Alan C. Sartorelli1

1Departments of Pharmacology and 2Internal Medicine, 3Section of Comparative Medicine, and Developmental Therapeutics Program, Cancer Center, Yale University School of Medicine, New Haven, CT

The impact of mouse multidrug resistance genes mdr1a/b and mrp1 on age-related differences in the toxicity and biodistribution of vincristine (VCR) was evaluated in wild-type, mrp1(-/-), mdr1a/b(-/-), and combined mdr1a/b(-/-), mrp1(-/-) weanling and adult mice given a single IP dose of VCR ranging from 0.0625 to 6 mg/kg. Weanling mice of all four genotypes were more sensitive than adult animals as determined by survival rate, average time of death, and pathologic findings. Wild-type animals were the least sensitive and combined\mdr1a/b(-/-), mrp1(-/-) mice the most sensitive to VCR toxicity. Mdr1a/b(-/-) and mrp1(-/-) genotypes exhibited intermediate sensitivities, with mdr1a/b(-/-) mice being more sensitive than mrp1(-/-) animals to the vinca alkaloid. Administration of [3H]VCR to wild-type and mdr1a/b(-/-), mrp1(-/-) animals revealed relatively greater accumulation of radioactive VCR equivalents in weanlings over adults in several tissues, with weanling mdr1a/b(-/-), mrp1(-/-) lung and heart exhibiting the greatest enhanced accumulation of 26- and 15-fold over adults, respectively. A similar cardiopulmonary differential accumulation of VCR was not observed in wild-type weanlings to adults. Semiquantitative RT-PCR expression analyses of ABC transporter genes in weanling and adult tissues of wild-type and combined mdr1a/b(-/-), mrp1(-/-) mice did not reveal major age-related differences in these ABC transporters that would explain the relatively greater toxicity observed in weanling mice. However, the greater cardiopulmonary accumulation of VCR equivalents seen in the combined mdr1a/b(-/-), mrp1(-/-) weanlings over that of adults underscores the potential for unique organ and age-related toxicities of this agent in the setting of transporter deficiency.

Key words: Vincristine toxicity; P-Glycoprotein; Mrp1; Multidrug resistance

Address correspondence to Alan C. Sartorelli, Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520. Tel: (203) 785-4533; Fax: (203) 703-2045; E-mail: alan.sartorelli@yale.edu

*These authors contributed equally to this work.

Oncology Research, Volume 14, pp. 345-354
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Copyright © 2004 Cognizant Comm. Corp.
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The Cellular Response to PPARg Ligands Is Related to the Phenotype of Neuroblastoma Cell Lines

Tiziana Servidei,1,2* Roberta Morosetti,2* Cristiano Ferlini,3 Gabriella Cusano,2 Giovanni Scambia,4 Renato Mastrangelo,2 and H. Phillip Koeffler5

1Molecular Biology Laboratory, 2Division of Pediatric Oncology, and 3Department of Obstetrics and Gynecology, Catholic University of Rome, Rome, Italy
4Centro di Ricerca e Formazione ad Alta Tecnologia delle Scienze Biomediche, Catholic University, Campobasso, Italy
5Division of Hematology/Oncology, Cedars-Sinai Medical Center, Los Angeles, CA

Neuroblastoma (NB) is a phenotypically heterogeneous tumor, displaying cells of neuronal, melanocytic, or glial/schwannian lineage. This cellular heterogeneity is also present in vitro, where cells of neuroblastic (N)- or stromal (S)-type may be identified. Ligands of peroxisome proliferator-activated receptor g (PPARg) have been shown to inhibit growth in different tumor cell lines. The purpose of this study was to determine PPARg expression and the response to its ligands in NB cell lines with different phenotypes. We used eight NB cell lines with N-, mixed, and S-phenotype. PPARg expression was found in all NB cell lines, regardless of their phenotype. Mutational analysis and transactivation assays showed that PPARg is not mutated and remains functional in NB cells. Two PPARg ligands, 15-deoxy-D12,14-prostaglandin J2 (PGJ2) and rosiglitazone, inhibited growth of all cell lines, with PGJ2 being the most potent agent. PGJ2, but not rosiglitazone, induced arrest of the cells in the G2/M phase as well as apoptosis. The sensitivity to the two ligands appeared to be more related to the phenotype than PPARg expression, with the S-type cells being less sensitive than the N-type, partly because of their lower capability of undergoing apoptosis. No synergistic effect on growth inhibition was observed when all cell lines were co-treated with 9-cis retinoic acid (9-cis RA) and rosiglitazone. Our data indicate that PPARg expression and function are maintained in phenotipically different NB cell lines. Activation of PPARg by its synthetic ligands might have a therapeutic role in advanced NB.

Key words: Prostaglandin J2; Rosiglitazone; Neuroblastoma; Phenotype; Inhibition of growth; Apoptosis; Cell cycle

Address correspondence to Tiziana Servidei, Division of Pediatric Oncology, Catholic University of Rome, Largo A. Gemelli 8-00168 Rome, Italy. Tel: +39-06-3058203; Fax: +39-06-3052751; E-mail: tservidei@rm.unicatt.it

*These two authors contributed equally to this work.

Oncology Research, Volume 14, pp. 355-362
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Copyright © 2004 Cognizant Comm. Corp.
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Screening Novel, Potent Multidrug-Resistant Modulators From Imidazole Derivatives

Li-ming Chen,1 Xing-Ping Wu,1 Ji-wu Ruan,2 Yong-ju Liang,1 Yan Ding,1 Zhi Shi,1 Xiu-wen Wang,1 Lian-Quan Gu,2 and Li-wu Fu1

1Cancer Center and 2School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou 510060, P. R. China

The overexpression of P-glycoprotein (P-gp) by tumor cells results in multidrug resistance (MDR) to structurally unrelated anticancer drugs. Combined therapy with MDR-related cytotoxins and MDR modulators is a promising strategy to overcome clinical MDR. This study was designed to screen potent MDR modulators from imidazole derivatives. Cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The intracellular accumulation of doxorubicin (Dox) was detected by fluorescence spectrophotometry. The function of P-gp was examined by Rhodamine 123 accumulation detected with flow cytometry (FCM). Among imidazole derivatives, FG020326, FG020327, and FG020318 were found to possess three- to fourfold stronger reversal MDR activity than verapamil, a well-known positive MDR modulator. Imidazole derivatives significantly increased the Dox accumulation and inhibited P-gp function exhibited by the increase of Rhodamine accumulation in MDR cells. The fold reversal of MDR was relative with the increase of Rhodamine accumulation. FG020326, FG020327, and FG020318 showed potent MDR reversal activity in vitro. Their mechanism of MDR reversal is associated with the inhibition of P-gp function and the increase of anticancer accumulation. These results suggest FG020326, FG020327, and FG020318 are promising to further study and develop.

Key words: Multidrug resistance; P-glycoprotein; Imidazole; Doxorubicin; Vincristine

Address correspondence to Liwu Fu, Cancer Center, Sun Yat-Sen University, Guangzhou 510060, China. Tel: +86-(20)-873-431-63; Fax: +86-(20)-873-433-92; E-mail: Fulw@gzsums.edu.cn or Fuliwu@hotmail.com

Oncology Research, Volume 14, pp. 363-372
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Copyright © 2004 Cognizant Comm. Corp.
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Apoptosis of Human T-Cell Acute Lymphoblastic Leukemia Cells by Diphenhydramine, an H1 Histamine Receptor Antagonist

Shawkat-Muhialdin Jangi, Aintzane Asumendi, Jon Arlucea, Naiara Nieto, Gorka Perez-Yarza, María C. Morales, María de la Fuente-Pinedo, and María D. Boyano

Department of Cell Biology and Histology, Faculty of Medicine and Dentistry, University of the Basque Country, Leioa E-48940, Vizcaya, Spain

Recently, it has been demonstrated that histamine plays an important role in the proliferation of normal and malignant cells. We have examined the effects of histamine, diphenhydramine, and cimetidine (H1 and H2 histamine receptor antagonists, respectively) on the in vitro proliferation of two human T-cell acute lymphoblastic leukemia cell lines, namely CCRF-CEM and Jurkat. Exogenous histamine did not alter the proliferation or viability of these cells. In contrast, diphenhydramine induced apoptosis in a dose- and time-dependent manner in both cell lines, whereas cimetidine failed to induce significant effects at similar concentrations. Diphenhydramine-induced apoptosis was evaluated in terms of morphology, flow cytometry, and the release of cytochrome c to the cytosol. The latter was partially mitigated by Bcl-2 overexpression. In human peripheral blood mononuclear cells, diphenhydramine inhibited cell proliferation without inducing apoptosis. Our findings indicate that endogenous histamine may be an important factor for the survival of CCRF-CEM, Jurkat, and peripheral blood mononuclear cells, and point to the potential application of H1 receptor antagonists as cytotoxic agents for the specific treatment of certain types of leukemia.

Key words: Histamine antagonist; Diphenhydramine; Cimetidine; Apoptosis; Mitochondrion

Address correspondence to María D. Boyano, Department of Cell Biology and Histology, Faculty of Medicine and Dentistry, University of the Basque Country, Leioa E-48940, Vizcaya, Spain. Tel: +34-94-601-5689; Fax: +34-94-464-8966; E-mail: gcpbolom@lg.ehu.es

Oncology Research, Volume 14, pp. 373-379
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Copyright © 2004 Cognizant Comm. Corp.
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Status of Methylthioadenosine Phosphorylase and its Impact on Cellular Response to L-Alanosine and Methylmercaptopurine Riboside in Human Soft Tissue Sarcoma Cells

WeiWei Li,1 Dan Su,1 Hiroo Mizobuchi,2 Daniel S. Martin,1 Bin Gu,1 Richard Gorlick,2 Peter Cole,1 and Joseph R. Bertino1

1Program of Molecular Pharmacology and Chemistry, 2Department of Pediatrics, Memorial Sloan-Kettering Cancer Center, New York, NY 10021

The aim of this study was to examine the expression of methylthioadenosine phosphorylase (MTAP) in 21 fresh tumor samples from patients with soft tissue sarcomas (STS) and 11 human soft tissue sarcoma cell lines, and to determine if loss of expression of this enzyme was correlated with increased sensitivity to L-alanosine and/or 6-methylmercaptopurine. We used a polyclonal antibody to measure the expression of MTAP in soft tissue sarcoma cell lines and in fresh tumor samples. Transfection of the HT-1080 cell line with a plasmid containing the cDNA for the MTAP gene was also performed to generate cell lines for in vitro and in vivo comparative sensitivity studies. MTAP was not expressed in 8 of 21 fresh STS tumors. The expression of MTAP was also not detectable in 3 of the 11 soft tissue sarcoma cell lines (HT-1080, HS42, and M-9110). These three cell lines were more sensitive to L-alanosine, a potent inhibitor of de novo AMP synthesis, and to an inhibitor of de novo purine nucleotide synthesis, 6-methylmercaptopurine riboside (MMPR). The IC50 values for L-alanosine and MMPR were >20-fold lower in MTAP-deficient cells than in MTAP-positive cells. Restoration of MTAP into HT-1080 MTAP-deficient cells also led to decreased sensitivity to L-alanosine and MMPR. An in vivo study using HT-1080 cell tumors with and without MTAP expression confirmed that tumors lacking MTAP were more sensitive to L-alanosine than tumors expressing MTAP. These results provide the basis for selective therapy using inhibitors of de novo purine nucleotide synthesis such as L-alanosine or MMPR to treat patients with STS lacking this enzyme.

Key words: Methylthioadenosine phosphorylase (MTAP); Drug sensitivity; L-Alanosine; Methylmercaptopurine riboside (MMPR); Soft tissue sarcoma cells

Address correspondence to Joseph R. Bertino at his current address: Cancer Institute of New Jersey, Robert Wood Johnson Medical School, New Brunswick, NJ 08901. Tel: (732) 325-8510; Fax: (732) 235-8181; E-mail: bertinoj@umdnj.edu

Oncology Research, Volume 14, pp. 381-386
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Copyright © 2004 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Differential Effects of Doxorubicin and Docetaxel on Nitric Oxide Production and Inducible Nitric Oxide Synthase Expression in MCF-7 Human Breast Cancer Cells

Gulperi Oktem,1 Bulent Karabulut,2 Nur Selvi,2 Canfeza Sezgin,2 Ulus Ali Sanli,2 Ruchan Uslu,2 Mine Ertem Yurtseven,1 and Serdar Bedii Omay2

1Histology and Embryology and 2Hematology-Oncology, Ege University Medical Faculty, Bornova 35100, Izmir, Turkey

Anthracyclines and docetaxel are frequently used agents in the chemotherapy of breast cancer. In this study we examined the role of inducible nitric oxide synthase (iNOS) expression during the cytotoxicity of these drugs in the MCF-7 breast cancer cell line. Cell viability and cytotoxicity were evaluated by the trypan blue dye exclusion method. Apoptosis and necrosis were determined by the acridine orange/ethidium bromide dye method. The percentage of apoptotic cells was significantly higher with doxorubicin. However, total cytotoxic cell numbers were higher in the docetaxel group compared with doxorubicin, with respect to the control. Most of the cells were seen to be necrotic with the dye method. Cell extracts during the apoptotic process were applied to immunoblot by anti-iNOS monoclonal antibodies. While there was an increase in iNOS expression during docetaxel induced-cytotoxicity, a significant decrease in iNOS expression was detected during doxorubicin-induced cytotoxicity. The Griess method was used for detection of nitrate levels. It was compatible with immunoblot results. These data open a window for further studies to understand the mechanism underlining the cytotoxicity of docetaxel and doxorubicin.

Key words: MCF-7; Inducible nitric oxide synthase (iNOS); Nitric oxide; Cytotoxicity; Doxorubicin; Docetaxel

Address correspondence to Serdar Bedii Omay, M.D., Ph.D., Ege University School of Medicine Department of Hematology-Oncology, 35100 Bornova, Izmir, Turkey. Tel/Fax: +90 232 374 73 21; E-mail: omay@med.ege.edu.tr

Oncology Research, Volume 14, pp. 387-397
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Copyright © 2004 Cognizant Comm. Corp.
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Inhibitory Effect of Topical Applications of Nondenatured Soymilk on the Formation and Growth of UVB-Induced Skin Tumors

Mou-Tuan Huang,1 Jian-Guo Xie,1 Connie B. Lin,2 Menas Kizoulis,2 Miri Seiberg,2 Stanley Shapiro,2 and Allan H. Conney1

1Susan Lehman Cullman Laboratory for Cancer Research, Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854-8020
2The Johnson & Johnson Skin Research Center, CPPW, 199 Grandview Road, Skillman, NJ 08558

Treatment of female SKH-1 hairless mice with ultraviolet B light twice a week for 20 weeks resulted in a population of tumor-free mice with a high risk of developing skin tumors during the next several months in the absence of additional UVB treatment (high-risk mice). Topical applications of nondenatured soymilk but not heat-denatured soymilk once a day, 5 days a week to these high-risk mice inhibited the formation and growth of skin tumors. Similar topical applications of soybean trypsin inhibitor or Bowman-Birk inhibitor also inhibited the formation and growth of skin tumors, but these agents were less active than nondenatured soymilk. Treatment of miniswine skin with nondenatured soymilk once a day for 5 days prior to UVB irradiation reduced or completely eliminated UVB-induced formation of thymine dimers and apoptotic cells in the epidermis. These data suggest that nondenatured soymilk could be applied to humans to prevent sunlight-induced skin damage and to reduce the risk of skin tumor formation and progression.

Key words: Cancer chemoprevention; Nondenatured soymilk; Serine protease inhibitors; Topical application

Address correspondence to Allan H. Conney, Susan Lehman Cullman Laboratory for Cancer Research, Department of Chemical Biology, Ernest Mario School of Pharmacy, The State University of New Jersey, 164 Frelinghuysen Road, Rutgers, Piscataway, NJ 08854-8020. Tel: (732) 445-4940; Fax: (732) 445-0687; E-mail: aconney@rci.rutgers.edu

Oncology Research, Volume 14, pp. 399-405
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Copyright © 2004 Cognizant Comm. Corp.
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Evidence That General Genomic Hypomethylation and Focal Hypermethylation Are Two Independent MolecularEevents of Non-Hodgkin's Lymphoma

Janelle T. Pini,1 Maria Franchina,1* Jeremy M. E. Taylor,2 and Peter H. Kay1

1Division of Pathology, School of Surgery and Pathology, The University of Western Australia, Nedlands, Western Australia, Australia 6907
2The Western Australian Centre for Pathology and Medical Research, Nedlands, Western Australia, Australia 6009

Changes in the DNA methylation profile, including general genomic hypomethylation and regional hypermethylation, have been shown to coexist in many neoplastic tissues. However, the relationship between, and significance of, these different forms of DNA methylation dysregulation in disease onset, progression, or maintenance remains unclear. Previously, our work has shown that the CpG dinucleotide-rich gene Myf-3 is hypermethylated in most cases of malignant lymphoproliferative disease (LPD). However, it is unknown whether malignant transformation of lymphoid cells is associated with general genomic hypomethylation and whether regional hypermethylation is restricted to CpG islands. The relationship between the status of general genomic methylation and the methylation of CpG and non-CpG islands can be clearly investigated in DNA from tumors of patients suffering malignant LPD, as monoclonalilty of malignant cells in LPD can be readily confirmed. In this study, the relationships between the methylation status of a region of the PAX7 paired box, which is not contained within a CpG island, general genomic hypomethylation, and the methylation status of Myf-3 was examined in 24 cases of LPD. Results revealed that hypermethylation of the PAX7 paired box is strongly associated with hypermethylation of Myf-3, indicating the abnormal hypermethylating activity in malignant lymphoid cells does not specifically target CpG islands. Further, general genomic hypomethylation was shown to be associated with malignant LPD but not with regional hypermethylation, indicating that the mechanisms responsible for the generation of each of these disturbed DNA methylation phenotypes act independently as one of a number of permissive but not essential steps in the malignant transformation of lymphoid cells.

Key words: Genomic hypomethylation; Hypermethylation; Myf-3; PAX7; Malignant lymphoproliferative disorders

Address correspondence to Janelle T. Pini, Molecular Pathology Laboratory, Division of Pathology, School of Surgery and Pathology, The University of Western Australia, Nedlands, WA, Australia 6907. Tel: (08) 9346 3455; Fax: (08) 9346 2891; E-mail: nellpini@cyllene.uwa.edu.au

*Present Address: Centre for Medical Research, The Western Australian Institute for Medical Research, The University of Western Australia, Western Australia, Australia 6907