The melanoma cell line FO-1 does not express HLA class I antigens and does not acquire them on the cell surface after incubation with IFN--y. Immunochemical studies showed that FO-1 cells synthesize HLA class I heavy chain, but do not synthesize t#2-microglobulin (02-it). The latter abnormality is associated with lack of 82-mRNA which remains undetectable in FO-1 cells incubated with IFN-'y. The defect was identified as a genetic lesion in the B2m gene, since DNA hybridization analysis detected a deletion of the first exon ofthe 5'-flanking region, and of a segment of the first intron of the B2m gene. HLA class I antigen expression was reconstituted on melanoma cells FO-1 after transfection with the wild-type mouse B2m gene, thereby confirming the abnormality of the endogenous B2m gene.The defect identified in FO-1 cells is distinct from that underlying the lack of HLA class I antigen expression by lymphoblastoid cells Daudi, but is remarkably similar to that causing lack of H-2 class I antigen expression by mouse lymphoblastoid cells R1 (TL-). These results suggest that genetic recombination in the 5' region of the B2m gene is a recurrent mechanism in B2m gene defects. In addition to contributing to our understanding of molecular abnormalities in HLA class I antigen expression by melanoma cells, FO-1 cells represent a useful model for analyzing the role of HLA class I antigens in the biology of melanoma cells and in their interaction with cells of the immune system. (J. Clin. Invest. 1991. 87:284-292.)
The effect of L-carnitine and some of its acyl derivatives on serum TNF production and lethality in a murine experimental endotoxin shock model was investigated. In some instances, serum IL-6 production was also evaluated. In this experimental model, C57BL/6 mice received 30 mg/kg LPS (E. cell 055:B5) injected intraperitoneally, while L-carnitine and its derivatives were administered according to different schedules. Serum levels of TNF and IL-6 were evaluated 1 h following LPS injection. The treated animals were also monitored daily for differences in body temperature, feeding, and survival for 10 days after LPS injection. Although some derivatives were able to significantly affect TNF production, the marked decrease in serum TNF levels of LPS-treated mice was not paralleled by a substantial increase in survival.
Testing of a panel of cultured human melanoma cells with radiolabelled anti-HLA-class4 monoclonal antibodies (MAbs) in a binding assay has shown lack of reactivity of FO-I and SK-MEL-33 cells and low reactivity of SK-MEL-I9 cells. SDS-PAGE analysis of the components immunoprecipitated from the 3 intrinsically radiolabelled melanoma cell lines by antibodies to the 2 subunits of HLA-class4 antigens has not detected p2-p in the immunoprecipitates from melanoma cells FO-I and SK-MEL-33 and only a low level of HLA-class4 heavy chain in the immunoprecipitate from SK-MEL-19 cells.Northern blotting analysis with probes specific for HLA-class4 heavy chain and for p2-p indicates that the abnormalities in HLA-class-I-antigen expression reflects a defect at the transcriptional level in The potential role of the immune system in the pathogenesis and clinical course of melanoma (Balch et al., 1989) has stimulated interest in the characterization of HLA-class4 and class-11-antigen expression by melanoma cells. Immunohistochemical staining of a large number of surgically removed melanoma lesions has shown reduction or loss of HLA class-I-antigen expression in about 10% of the primary lesions and in about 30% of metastatic lesions (Ruiter et al., 1991). Because of the role of HLA-class-I antigens in the recognition of target cells by cytotoxic T cells (McMichael, 1980) and by NK cells (Ljunggren and Karre, 1990), these changes are likely to affect the interaction of melanoma cells with the host's immune system. The potential clinical significance of reduced HLAclass-I-antigen expression by melanoma cells is suggested by the statistically significant, although not absolute, association between low level of HLA-class-I antigens in melanoma lesions and poor clinical course of the disease (Van Duinen et al., 1988). The molecular abnormalities underlying the defects in HLA-class-I-antigen expression by melanoma cells have been characterized only to a limited extent (Versteeg et al., 1988; D'Urso et al., 1991), although this information contributes to our understanding of the regulatory mechanisms controlling HLAclass-I-antigen synthesis and membrane insertion and is required to develop approaches to correct reduction or lack of HLA-class-I-antigen expression by melanoma cells. The latter may have therapeutic implications, if abnormalities in the HLA-class-I-antigen expression are proven to play a role in the clinical course of the disease.In previous reports we have described the expression of HLA-class-I and class-I1 antigens by melanoma cell lines in long-term culture and in surgically removed melanoma lesions. In the present report, we describe the molecular defects which underlie abnormalities in HLA-class-I-antigen expression by 3 melanoma cell lines. MATERIAL AND METHODSThe growth conditions of cell lines, the characteristics of monoclonal and polyclonal antibodies and the methodology to perform serological and immunochemical assays and RNA hybridization analysis have been described elsewhere (D'Urso et al., 1991). RESULTSTestin...
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