ABSTRACT

Calcium-binding proteins may endow tumor cells with properties related to their malignancy and metastatic phenotype. Chromatographic procedures and amino acid sequence analysis were used in this study to identify seven calcium-binding proteins, annexin VI, cap g, annexin V, calmodulin, S100A11, S100B and S100A6, associated with uveal melanoma, the primary ocular tumor of adults. This series of calcium-binding proteins was identified in both primary tumors and cell lines of uveal melanoma. Several of the proteins were shown by immunochemical methods to be differentially expressed between normal uveal melanocytes and malignant melanomas of the uvea. In addition, the expression of S100A6 may correlate with the malignant properties of the tumor.

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To determine which of the N-acetyltransferase (NAT) alleles [monomorphic (NAT1) or polymorphic (NAT2)] are expressed in the target cells for arylamine carcinogenesis, namely normal human uroepithelial cells, cDNA was prepared from cellular RNA and amplified by polymerase chain reaction (PCR), using upstream primer 1 comprising the 5' end (nt 47-68) and either downstream primers 2 (nt 908-889) or 3 (nt 953-931) corresponding with the 3' end. With primers 1 and 2, selective for NAT1, a characteristic 861 bp DNA fragment was obtained, whereas with primers 1 and 3, selective for NAT2, a characteristic 907 bp fragment was formed. Similarly, the PCR-amplified cDNA products from the SV40-immortalized human uroepithelial cell line were also found to contain both NAT1 and NAT2. Restriction fragment length polymorphism (RFLP) analysis with HincII (digesting NAT2 to produce 659 bp and 248 bp fragments) and HindIII (digesting NAT1 to produce a 786 bp fragment) further confirmed the authenticity of the NAT alleles. Furthermore, the NAT genotypes of 38 individuals were determined by PCR amplification of lymphocyte DNA and subsequent RFLP analysis using TaqI, KpnI and BamHI. The genotypes were compared to their in vivo acetylator phenotypes which were determined by measuring 5-acetylamino-6-formylamino-3-methyluracil and 1-methylxanthine in urine following administration of caffeine. A good correlation between the genotype and phenotype was obtained in the study population and the frequency of NAT2 allele distribution was M1 > wild-type > M2 > M3. These results suggest that susceptibility to arylamine-induced bladder cancer might be influenced by both hepatic and bladder NAT and that the NAT genotype might be a useful biomarker for screening high risk individuals for bladder cancer resulting from exposure to arylamines.

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