Evaluation of oxidative damage and inhibition of DNA repair in an in vitro study of nickel exposure.

It has been suggested that Nickel is involved in oxidative damage and inhibition of DNA repair. We studied the effects of NiSO4 on oxidative stress and DNA repair in Jurkat cells to elucidate its mechanism of action. Cells were treated with H2O2 and ROS generation (by flow cytometry), and oxidative DNA damage (as tail moment by Fpg-enzyme comet test), were evaluated immediately and after 4 and 24 h of DNA damage recovery occurred in presence or absence of NiSO4 (0.017 and 0.17 microM) to clarify possible interactions of Ni with DNA repair processes. Moreover, cells were exposed to the same doses of NiSO4 for 4 and 24 hours to evaluate its direct oxidative effect. The results of the comet test showed high tail moment immediately after oxidative burst with a decreasing after 4 h of DNA recovery, and a slight increase after 24 h of recovery. The decreases were more limited for cells treated with NiSO4 0.17 microM indicating an inhibition of oxidative DNA damage repair by this substance. An induction of ROS was observed after 4 h of incubation with higher dose of NiSO4. Cells treated with H2O2 showed the highest level of ROS after 4 h of recovery in presence of NiSO4 0.17 microM that remained at elevated levels also after 24 h of recovery suggesting a synergistic action of Ni with H2O2 in the reduction of cellular anti-oxidative defence activities.

Low prevalence of selective human leukocyte antigen (HLA)-A and HLA-B epitope losses in early-passage tumor cell lines.

The down-regulation of human leukocyte antigen (HLA) class I molecules, especially the selective down-regulation of certain allelic products, is believed to represent a major mechanism of tumor escape from immune surveillance. In the present report, an original approach is described to precisely evaluate and classify HLA class I epitope losses in 30 cancer patients with malignant melanoma and lung, breast, endometrium, ovary, and colon carcinoma tumors. Early-passage tumor cell lines were established in culture from the corresponding metastatic tumor lesions obtained in each patient. Both the cell lines and the tumor lesions were compared, in their HLA-A and -B expression, to the peripheral blood mononuclear cells (PBMCs) obtained from the same patient (autologous PBMCs). On the basis of HLA-genotyping data, the appropriate monoclonal antibodies identifying mono- and poly-morphic HLA-A and HLA-B epitopes were selected from a panel of 34 antibodies for a total of 24 testable alleles. The selected antibodies were used not only in immunohistochemical assays on cryostatic tumor sections and cytospins of PBMCs but also in quantitative, sensitive flow cytometry assays on early-passage tumor cells and PBMC suspensions. With this latter method, a low overall HLA expression was detected in 26 tumor cell explants and a complete, generalized HLA-A, HLA-B, HLA-C loss in the remaining 4 cases. However, no complete, selective loss of any of the 45 tested HLA-A and HLA-B allomorphs was observed. Sequences from all of the HLA class I alleles could be detected at the genomic DNA level in tumor cells and tissues. At variance from the literature and the results of immunohistochemical experiments performed in parallel on the corresponding tumor lesions, the relative proportions of the various HLA epitopes were relatively preserved in each early-passage cell line/PBMC pair, and selective increases, rather than decreases, in the expression of polymorphic HLA epitopes had the highest prevalence and greatest magnitude. Our data suggest an alternative tumor stealth strategy in which up- and down-regulation are equally important. This alternative model of tumor-host interaction better fits the available models of tumor cell recognition by CTLs and natural killer cells bearing activatory and inhibitory receptors for HLA-A, HLA-B, HLA-C molecules.

Biosynthesis of HLA-C heavy chains in melanoma cells with multiple defects in the expression of HLA-A, -B, -C molecules.

Recent investigations have shown that malignant transformation may down-regulate the expression of class I HLA molecules, beta2-microglobulin (beta2m) and members of the antigen-processing machinery. In the present study, we HLA-genotyped and identified at a biochemical level the three (HLA-A25, -B8, -Cw7) class I alleles expressed by the previously described [D'Urso CM et al (1992) J Clin Invest 87: 284-292] beta2m-defective human melanoma FO-1 cell line and tested their ability to interact with calnexin, calreticulin and the TAP (transporter associated with antigen processing) complex. All these alleles were found to bind calnexin, but not calreticulin or the poorly expressed TAP complex, both in parental and beta2m-transfected FO-1 cells, demonstrating a complex defect of class I expression in FO-1 cells. In these conditions, Cw7 heavy chains interacted with calnexin more strongly than A25 and B8, and preferentially accumulated in the endoplasmic reticulum, in both a calnexin-associated and a calnexin-free form. In addition, they could be transported to the cell surface at low levels even in the absence of beta2m, without undergoing terminal glycosylation. These results establish a parallel between HLA-C and the murine Db and Ld molecules which have been found to be surface expressed and functional in beta2m-defective cells. They also demonstrate distinctive features of HLA-C molecules. We propose that the accumulation of several assembly intermediates of HLA-C might favour the binding of peptide antigens not readily bound by HLA-A and -B molecules in neoplastic cells with suboptimal class I expression.

HLA-A, -B, -C genotyping and expression in human nonlymphoid tumor cell lines.

A combination of molecular genotyping and protein biochemistry methods was used to assess the HLA-A, -B, -C genotyping and expression of six tumor cell lines. Four cell lines had been previously HLA typed by conventional serologic methods. Two could not be typed by serology because deficient in the surface expression of HLA-A, -B, -C molecules. As shown herein, all the 25 alleles carried by the six tested cell lines were typed at the DNA level. In addition, discrepancies between the previous serologic and the present DNA typing results were detected in 9 of the 21 tested serologic specificities. Typing at the protein level by isoelectric focusing and allele-specific monoclonal antibodies confirmed the DNA typing data. Our results exemplify the limits of the serologic typing procedures and demonstrate that molecular methods are highly desirable to conduct functional experiments and identify HLA losses in neoplastic cells at single allele level.

Conformation and surface expression of free HLA-CW1 heavy chains in the absence of beta 2-microglobulin.

A beta 2-microglobulin (beta 2m)-deficient kidney carcinoma cell line and three monoclonal antibodies to the alpha 1 (L31), alpha 2 (W6/32), and alpha 3 (Q1/28) domain of class I HLA molecules were selected to assess the role of beta 2m in regulating the conformation and surface expression of HLA-C molecules. HLA-A2, -B27, and -CW1 molecules synthesized by beta 2m-deficient cells were compared to heavy chains synthesized in transfectants expressing a large excess of beta 2m. As assessed by differential binding with monoclonal antibodies and partitioning studies in the detergent TX-114, no HLA-A2, -B27, or -CW1 molecules can be expressed, in a correct conformation, by beta 2m-deficient cells. These cells, however, do express low but significant amounts of free HLA-CW1 heavy chains at the cell surface. Transfection with beta 2m causes a coordinate change in the antibody reactivity of the three domains of HLA-CW1 molecules, thereby providing the first experimental demonstration that assembly with beta 2m affects the folding of not only the alpha 1 and alpha 2, but also of the alpha 3 domain. HLA-CW1 heavy chains, when free of beta 2m, are less soluble in the detergent TX-114 than free HLA-B27 heavy chains, and when associated with beta 2m share an alpha 3 domain epitope with free HLA-A2 and -B27 heavy chains. Moreover, their assembly with beta 2m is largely incomplete. Those data additionally demonstrate an impaired ability of HLA-CW1 to properly fold and establish a close similarity of HLA-CW1 to murine Db and Ld molecules. Although the functional role, if any, of free HLA-CW1 heavy chains remains to be determined, the present study demonstrates that the absence of beta 2m does not completely ablate class I expression in neoplastic cells of human origin.

Distinctive features of the alpha 1-domain alpha helix of HLA-C heavy chains free of beta 2-microglobulin.

Only a few monoclonal antibodies are available with a restricted specificity to HLA-C products. In the present report, we demonstrate that antibody L31, previously shown to react with beta 2m-less (free) class I MHC heavy chains, binds to an epitope (residues 66-68 of the alpha 1 domain alpha helix) present on all the HLA-C alleles corresponding to the accepted (CW1 through CW8) serologic specificities, and on a few HLA-B heavy chains sharing with HLA-C an aromatic residue at position 67. Extensive IEF blot testing of HLA homozygous, EBV-transformed B-lymphoid cells indicates that HLA-C molecules are present at significantly lower levels than HLA-B polypeptides not only at cell surface, as previously demonstrated, but also in total cellular extracts. Testing of metabolically labeled HLA-CW1, -CW5, and -CW6 transfectants and HLA homozygous lymphoid cells, particularly HLA-CW1-expressing cells, demonstrates that the L31 epitope is present on a subpopulation of naturally occurring HLA-C molecules distinct from that identified by antibody W6/32 to beta 2m-associated heavy chains. Pulse-chase experiments demonstrate that this epitope is transiently made available to antibody binding at early biosynthetic stages, but becomes hidden upon assembly with beta 2m. Thus, free HLA-C and other Y/F67+ heavy chains are characterized by distinctive antibody binding features in a region (residues 66-68) included in a previously identified HLA-C restricted motif, which has been suggested to be the primary cause of distinctive features of the antigen-binding groove, low affinity for endogenous peptide antigens and beta 2m, and preferential uptake of exogenous peptides, possibly of viral origin. We also show that HLA-CW1 heavy chains, both free and beta 2m associated, acquire sialilation. Free HLA-CW1 heavy chains are expressed at the cell surface even when unsialilated, albeit at low levels.

DR alpha: E beta heterodimers in DRA transgenic mice hinder expression of E alpha: E beta molecules and are more efficient in antigen presentation.

HLA-DRA transgenic (tg) mice on H-2d background were constructed to study assembly, expression and function of DR alpha: E beta class II heterodimers when an alternate E alpha chain is available. Cytofluorimetric analysis and immunoprecipitation studies demonstrate that the majority (90%) of E beta d molecules on class II-positive splenocytes from DRA-tg mice are associated with DR alpha rather than E alpha chains. To characterize the functional role of the interspecies as compared with the wild-type I-E molecules, MHC restriction and T cell epitope immunodominance of synthetic peptides spanning the entire sequence of 65 kDa heat shock protein (hsp) from Mycobacterium tuberculosis were determined in hsp-primed DRA-tg and DBA/2 mice. A similar pattern of responsiveness was observed in both strains, but hsp epitopes recalled a higher response in DRA-tg as compared with DBA/2 mice. A panel of T cell hybridomas specific for two hsp peptides or a hen egg white lysozyme peptide presented by both DR alpha: E beta d and E alpha d: E beta d was studied in detail. Surprisingly, DR alpha: E beta d dimers present these peptides more efficiently than E alpha d: E beta d, even when the TCR was selected in mice expressing only E alpha d: E beta d molecules. The higher efficiency of antigen presentation by DR alpha: E beta d dimers does not appear to depend on increased binding affinity for peptides, as demonstrated by competition for antigen presentation, nor on increased efficiency in the interaction with CD4 molecules. Rather, the higher efficiency of antigen presentation could be explained by a more effective ligand-TCR interaction. This is consistent with molecular modeling based on the class II structure, indicating that 16 out of 17 substitutions between the first domain of E alpha d and DR alpha chains ile outside the peptide binding groove and are potentially available for interaction with the TCR.