SV40-transformed Cells Research Articles

ANG II is a mitogen for a murine cell line isolated from medullary thick ascending limb of Henle's loop.

A murine SV40-transformed renal epithelial cell line derived from medullary thick ascending limb of Henle's loop (MTAL) was established and characterized by morphology, antigen expression, and biochemical criteria. These MTAL cells express a single class of high-affinity receptors for angiotensin II (ANG II) and transcripts for the AT1 subtype of ANG II receptors. ANG II, in a dose-dependent manner, induced proliferation of MTAL cells. This observation is in striking contrast to syngeneic proximal tubular cells in which it was previously shown that the peptide induced cellular hypertrophy and slightly inhibited proliferation [G. Wolf and E. G. Neilson. Am. J. Physiol. 259 (Renal Fluid Electrolyte Physiol. 28: F768-F777, 1990]. The AT1-receptor antagonist losartan (10(-6) M), but not an AT2-receptor antagonist, blocked the mitogenic effects of ANG II in MTAL cells. Coincubation of quiescent MTAL cells with ANG II and 5% fetal calf serum further increased proliferation compared with cells grown only in serum. In contrast to proximal tubular cells, ANG II failed to induce transforming growth factor-beta 1 mRNA and protein synthesis in MTAL cells. Our data collectively suggest that ANG II is a mitogen for MTAL cells in vitro. Therefore, epithelial cells derived from different parts of the nephron, even when transformed with SV40 virus and while under cell culture conditions, exhibit a distinct pattern of growth behavior after stimulation with ANG II.

Development and characterization of a human marrow stromal cell line that enhances osteoclast-like cell formation.

We established a human bone marrow stromal cell line (Saka) by infecting marrow adherent cells from semisolid marrow cultures with a recombinant simian virus-40 (SV40) virus. The cells expressed SV40 large tumor antigen, had a fibroblast-like shape, and expressed fibronectin and vimentin. They did not contain detectable alkaline phosphatase activity; express myeloid, lymphoid, or factor VIII-associated antigens; or develop adipocyte-like characteristics with dexamethasone treatment. Polymerase chain reaction analysis of Saka cell RNA detected expression of messenger RNAs for interleukin-6 (IL-6), IL-1 beta, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, stem cell factor, and the 1,25-dihydroxyvitamin D3 receptor. Coculture of Saka cells with human marrow mononuclear cells enhanced formation of osteoclast-like multinucleated cells (MNC) in long term human bone marrow cultures. These MNC expressed calcitonin receptors and formed resorption lacunae on dentine. In contrast, coculture of marrow mononuclear cells with other SV40-transformed human marrow stromal cell lines did not increase MNC formation. Conditioned medium from Saka cells or coculture of bone marrow and Saka cells separated by a Millipore membrane did not enhance MNC formation. Addition of a neutralizing antibody to IL-6 or IL-1 beta blocked the effects of Saka cells on MNC formation. These results suggest that marrow stromal cells enhance osteoclast formation in part through direct cell to cell contact and production of IL-6 and/or IL-1 beta.

Characterization of factors regulating successful immunotherapy using a tumor‐specific cytotoxic T lymphocyte clone: Role of interleukin‐2, cycling pattern of lytic activity and adhesion molecules

Adoptive immunotherapy against cancer has met with varying degrees of success, the reasons for which remain unclear. The present study characterizes factors that regulate successful immunotherapy of mice bearing a syngeneic T-cell lymphoma, designated LSA, using a tumor-specific cytotoxic T lymphocyte (CTL) clone, PE-9. Adoptive transfer of PE-9 cells afforded significant protection in normal but not in nude mice against LSA tumor. However, the PE-9 cells could protect the nude mice when injected along with normal CD4+ T cells. Administration of IL-2 along with PE-9 cells failed to enhance tumor immunotherapy. IL-2 therapy was toxic inasmuch as injection of the CTL clone PE-9 + IL-2, but not PE-9 or IL-2 alone, for 5 days into irradiated mice caused vascular leak syndrome (VLS). PE-9 cells cultured with high doses of rIL-2 in vitro also caused TCR-independent and MHC-unrestricted lysis of SV40-transformed endothelial cells. Furthermore, PE-9 cells cultured in vitro for 24-96 hr with IL-2 exhibited cycling pattern of tumor-specific cytotoxicity with maximum cytotoxicity demonstrable at 48 hr and virtually no cytolytic activity at 96 hr of culture or thereafter. The loss of cytotoxicity correlated with down-regulation of several adhesion molecule expressions on PE-9 cells, particularly the alpha beta-TCR, as well as the mRNA for TNF-alpha, IFN-gamma and perforin, although the levels of granzyme A were not altered. Interestingly, the outcome of immunotherapy by PE-9 cells depended on the cycling pattern of cytotoxicity. Our data suggest that successful immunotherapy against cancer using a CTL clone depends on several factors, such as the cycling pattern of lytic activity, density of adhesion molecules, levels of cytokines expressed and the ability of IL-2 and CTL to trigger VLS.

Phosphorylation studies on rat p53 using the baculovirus expression system. Manipulation of the phosphorylation state with okadaic acid and influence on DNA binding.

To elucidate the role of phosphorylation of p53 we used the baculovirus expression system to obtain high yields of protein eventually in distinct phosphorylation states. Initially, we obtained only marginal phosphorylation, despite high levels of expression. Two-dimensional phosphopeptide maps exhibited the same pattern as known from rat cells although some sites were underrepresented. Coexpression of simian virus 40 (SV40) large T antigen or cyclin-dependent kinases, cdc2 or cdk2, had only marginal effects on the phosphorylation state of p53. However, when we employed the phosphatase inhibitor okadaic acid, overall phosphorylation of p53 was drastically enhanced in a dose-dependent manner and resembled that of p53 from SV40-transformed rat cells. This hyperphosphorylation resulted in enhanced binding of a consensus oligonucleotide as revealed by electrophoretic mobility shift assays. To assess the role of individual phosphorylation sites, we generated a set of mutants at putative or identified sites. All mutants retained the ability to bind wild-type conformation-specific antibody Pab1620, to complex with SV40 large T antigen, and to bind to the consensus oligonucleotide. Moreover, most mutants exhibited enhanced DNA binding upon okadaic acid treatment, except for a mutant at the cdk site which failed to do so. These data show that: (a) insect cells contain all the protein kinases necessary for phosphorylation of a mammalian protein, p53; (b) in insect cells the ratio of kinase/phosphatase activities differs from that in mammalian cells so that underphosphorylation of recombinant proteins in this system may result from high phosphatase activities rather than saturation of kinases with recombinant substrate; (c) the system can be manipulated to obtain subpopulations of recombinant protein in a desired phosphorylation state, and (d) phosphorylation may regulate the DNA-binding activity of p53.

Phosphorylation Studies on Rat p53 Using the Baculovirus Expression System. Manipulation of the Phosphorylation State with Okadaic Acid and Influence on DNA Binding

To elucidate the role of phosphorylation of p53 we used the baculovirus expression system to obtain high yields of protein eventually in distinct phosphorylation states. Initially, we obtained only marginal phosphorylation, despite high levels of expression. Two-dimensional phosphopeptide maps exhibited the same pattern as known from rat cells although some sites were underrepresented. Coexpression of simian virus 40 (SV40) large T antigen or cyclin-dependent kinases, cdc2 or cdk2, had only marginal effects on the phosphorylation state of p53. However, when we employed the phosphatase inhibitor okadaic acid, overall phosphorylation of p53 was drastically enhanced in a dose-dependent manner and resembled that of p53 from SV40-transformed rat cells. This hyperphosphorylation resulted in enhanced binding of a consensus oligonucleotide as revealed by electrophoretic mobility shift assays. To assess the role of individual phosphorylation sites, we generated a set of mutants at putative or identified sites. All mutants retained the ability to bind wild-type conformation-specific antibody Pab1620, to complex with SV40 large T antigen, and to bind to the consensus oligonucleotide. Moreover, most mutants exhibited enhanced DNA binding upon okadaic acid treatment, except for a mutant at the cdk site which failed to do so. These data show that: (a) insect cells contain all the protein kinases necessary for phosphorylation of a mammalian protein, p53; (b) in insect cells the ratio of kinase/phosphatase activities differs from that in mammalian cells so that underphosphorylation of recombinant proteins in this system may result from high phosphatase activities rather than saturation of kinases with recombinant substrate; (c) the system can be manipulated to obtain subpopulations of recombinant protein in a desired phosphorylation state, and (d) phosphorylation may regulate the DNA-binding activity of p53.

Oxygen metabolites modulate sodium transport in gerbil middle ear epithelium: involvement of PGE2

The middle ear epithelium and respiratory epithelia share basic properties such as homeostasis of air-filled cavities and mucociliary clearance toward the pharynx. With the middle ear SV40-transformed (MESV) cell line, we used the short-circuit current (Isc) technique to investigate changes in ion transport induced by oxidants. Xanthine and xanthine oxidase on the basal side of the monolayers dramatically increased Isc up to 50%. This effect was not affected by superoxide dismutase or mannitol, but could be blunted by catalase or 1,3-dimethyl-2-thiourea. Increasing concentrations of H2O2 from 10(-5) to 5 x 10(-4) M produced a dose-dependent increase in Isc from 0.26 +/- 0.16 up to 4.21 +/- 0.43 microA/cm2 (P < 0.05, n = 5). Concentration of half-maximal stimulation (EC50) was 4.68 x 10(-5) M. This effect was inhibited by indomethacin and was related to a sodium transport, since the H2O2-induced increase in Isc could be prevented or abolished by 1) apical addition of benzamil (10(-6)M) and 2) substitution of sodium with N-methyl-glucamine. H2O2 exposure also induced indomethacin-sensitive increase in released prostaglandin (PG) E2 (EC50 = 5.62 x 10(-5) M) and in cAMP content (EC50 = 3.95 x 10(-5) M) with similar kinetics. These results suggest that exposure of MESV cells to oxidants stimulates the production of PGE2, which in turn increases the transepithelial sodium transport rate.

Monospecific Polyclonal Anti‐Anti‐Idiotypic Antibodies to the Carboxyterminal Undecapeptide of the SV40 Large Tumour Antigen

The murine monoclonal antibody PAb1605 defines an epitope, peptide Lys(698)-Thr(708) (KT), on the carboxyterminus of the tumour(T)antigen of SV40-transformed cells. In vivo and in vitro experiments had shown that this sequence represents an epitope for both humoral and cellular immune responses. When injected into rabbits PAb1605 induces anti-idiotypic antibodies (Ab-2). Ab-2 beta (internal image type) was purified by adsorption chromatography and characterized by the ability of KT to compete with the binding of ab-2 with ab-1. Murine anti-anti-idiotypic antibodies (ab-3) were obtained by immunization of mice with ab-2 beta. Both ab-1 and ab-3 JgG showed affinities to immunoprecipitated SV40 T antigen by immunoblot analysis and to nuclear SV40 T antigen by the immunofluorescence assay. The binding of ab-3 to SV40 T antigen was completely inhibited by competition with KT. We conclude that the polyclonal ab-3 is of the ab-3 subtype and specific for only one epitope which is represented by KT and defined by ab-1. The results demonstrate that the specificity for a defined peptide epitope of an antibody was conserved even after two consecutive steps of anti-idiotypic-antibody formation in two host species. Since this postulate of network theory could be verified for a sequence of a tumour-associated antigen which represents a B- and T cell epitope, this model is of great interest for further tumour immunological studies.

Spontaneous chromosomal aberrations in Fanconi anaemia, ataxia telangiectasia fibroblast and Bloom's syndrome lymphoblastoid cell lines as detected by conventional cytogenetic analysis and fluorescence in situ hybridisation (FISH) technique

Several primary and transformed human cell lines derived from cancer prone patients are employed routinely for biochemical and DNA repair studies. Since transformation leads to some chromosomal instability a cytogenetic analysis of spontaneous chromosome aberrations in fibroblast cell lines derived from patients with Fanconi anaemia (FA), ataxia telangiectasia (AT), and in lymphoblastoid cell lines derived from patients with Bloom's syndrome (BS), was undertaken. Unstable aberrations were analysed in Giemsa stained preparations and the chromosome painting technique was used for evaluating the frequencies of stable aberrations (translocations). In addition, the frequency of sister-chromatid exchanges (SCEs) was determined in differentially stained metaphases. The SV40-transformed fibroblasts from these cell lines have higher frequencies of unstable aberrations than the primary fibroblasts. In the four lymphoblastoid cell lines derived from BS patients higher frequencies of spontaneously occurring chromosomal aberrations in comparison to normal TK6wt cells were also evident. The frequency of spontaneously occurring chromosome translocations was determined with fluorescence in situ hybridisation (FISH) and using DNA libraries specific for chromosomes 1, 2, 3, 4, 7, 8, 11, 14, 19, 20 and X. The translocation levels were found to be elevated for primary FA fibroblasts and lymphoblastoid cells derived from BS patients in comparison with control cell lines, hetero- and homozygote BS cell lines not differing in this respect. The SV40-transformed cell lines showed very high frequencies of translocations independent of their origin and almost every cell contained at least one translocation. In addition, clonal translocations were found in transformed control TK6wt and AT cell lines for chromosomes 20 and 14, respectively. The spontaneous frequencies of SCEs were similar in transformed fibroblasts derived from normal individuals and AT patients, whereas in SV40-transformed FA cells these were higher (4-fold). Among cell lines derived from BS patients, heterozygote lines behaved like control, whereas in homozygote cell lines very high frequencies of SCEs (about 12-fold) were evident.

Unique and selective mitogenic effects of vanadate on SV40-transformed cells.

Vanadate and insulin both function as unique complete mitogens for SV40-transformed 3T3T cells, designated CSV3-1, but not for nontransformed 3T3T cells. The mitogenic effects induced by vanadate and insulin in CSV3-1 cells are mediated by different signaling mechanisms. For example, vanadate does not stimulate the tyrosine phosphorylation of the insulin receptor beta-subunit nor the 170 kDa insulin receptor substrate-1. Instead, vanadate induces a marked increase in tyrosine phosphorylation of 55 and 64 kDa proteins that is not observed in insulin-stimulated CSV3-1 cells. Perhaps most interestingly, vandate-induced mitogenesis is associated with the selective induction of c-jun and junB expression without significantly inducing c-fos or c-myc. Furthermore, treatment of CSV3-1 cells with genistein abolishes the effects of vanadate on protein tyrosine phosphorylation and c-jun induction. These and related data suggest that modulation of protein tyrosine phosphorylation and c-jun and junB expression may serve the critical roles in mediating vandate-induced mitogenesis in SV40-transformed cells.

Variations in tRNA modifications, particularly of their queuine content in higher eukaryotes. Its relation to malignancy grading

Literature references dealing with the variations in the modification level of nucleosides in total eukaryotic tRNAs as a function of different physiological status and after drug administration as well as in sequenced cytoplasmic tRNAs between normal and tumor cells and in SV40-transformed cells are reviewed. In addition, special attention is given to guanine replacement of queuine in the first position of the anticodon of tRNAs. A correlation between the level of this undermodification in cancer tissues and the malignancy grading could be found in human ovarian tumors, confirming the results reported in several laboratories for lymphomas and lung cancer tissues. Indeed tRNAs from primary and metastatic human ovarian malignant tumors are Q deficient as compared to tRNAs from normal tissues or benign tumors: thus queuine deficiency increases with malignancy and grading of differentiation.

FISH analyses of a newly established thyroid tumor cell line showing a t(1;19)(p35 or p36.1;q13) reveal that the breakpoint lies between 19q13.3-13.4 and 19q13.4.

Translocations involving the long arm of human chromosome 19 are specific chromosomal alterations found in a subset of benign thyroid tumors. In the present communication we describe the establishment of an SV40-transformed cell line derived from a thyroid adenoma with a t(1;19)(p35 or p36.1;q13). Cytogenetic studies were carried out on this cell line, using a series of cosmids mapping along the long arm of chromosome 19. In situ hybridization with probes for RYR1, ATP1A3, BCKDHA, ERCC1, POLD1, and TRPT revealed that only TRPT mapped distal to the breakpoint of the translocation. The results indicated that the breakpoint is located between POLD1 and TRPT at the boundary between chromosome bands 19q13.3 and 19q13.4.

ENHANCED KINASE-ACTIVITY IN SV40-TRANSFORMED CELLS MAY BE COMPENSATED BY ENHANCED PHOSPHATASE-ACTIVITY IN REVERTANTS AS REFLECTED BY PHOSPHORYLATION OF P53

Transformation of rat cells with SV40 large T antigen results in activation of protein kinases and hyperphosphorylation of the tumor suppressor protein p53. In searching for cellular targets involved in SV40-mediated transformation, flat revertants of SV40-transformed rat cells had been isolated carrying a presumptive defect in a cellular gene (Bauer et al: J Virol 61: 1821, 1987). In this investigation, we asked whether the phosphorylation state of p53 might be affected in the revertants. Two-dimensional phosphopeptide analyses revealed indeed a characteristic reduction of phosphorylation of p53 compared to the parental transformed cells. However, when we employed the phosphatase inhibitor okadaic acid in vivo hyperphosphorylation of p53 resumed indicating that the kinases involved in phosphorylation of p53 were fully active but counterbalanced by enhanced phosphatase activity. Indeed, the phosphate turnover of p53 in vivo and phosphatase activity towards p53 in vitro was higher in the revertants than in the parental transformants. These findings demonstrate that the transformation state of a cell correlates with the phosphorylation state of p53 which in turn can be regulated in different ways, enhanced kinase activity in transformed cells may be counteracted by enhanced phosphatase activity in revertant cells.

Characterization of spontaneous and induced mutations in SV40-transformed normal and ataxia telangiectasia cell lines.

Spontaneous and induced mutations at the HPRT locus were analyzed in one normal (MRC5CV1) and one ataxia telangiectasia (AT5BIVA) SV40-transformed cell line derived from male donors. Multiplex PCR and Southern analyses revealed a high frequency of spontaneous deletion mutations that may be a consequence of the SV40 transformation. Four mutagens (ethyl methanesulfonate, bromodeoxyuridine, bleomycin, adriamycin), which differ in their types of primary DNA lesions, caused specific patterns of mutations. By using fluorescence in situ hybridization (FISH) techniques, we were able to show that more than 90% of the AT5BIVA cells contained two X chromosomes with HPRT alleles, while in more than 90% of the MRC5CV1 cells genomic hemizygosity for the HPRT gene was found. Taking into account these findings we found that the AT5BIVA cell line possesses spontaneous hypermutability as well as hypersensitivity and hypermutability to bleomycin (BLM) and adriamycin (AM). Both mutagens induced deletion mutations in both cell lines, but more complex mutations and larger deletions were found in AT5BIVA cells.

Expression and phosphorylation of NHE1 in wild-type and transformed human and rodent fibroblasts.

Activation and phosphorylation of the Na+/H+ exchanger occurs with a diverse group of mitogens such as phorbol myristate acetate (PMA), epidermal growth factor (EGF), thrombin and serum (Sardet et al., 1990, Science, 247:723-726). Some of these growth factors have been shown to differentially activate the Na+/H+ exchanger in various fibroblasts (Etscheid et al., 1990, Am. J. Physiol., 259:C549-C556; Muldoon, L.L., et al., 1987, Am. J. Physiol., 253:C219-C229). However, alterations in the expression and phosphorylation of NHE1 in various fibroblasts has not been examined with respect to a potential mechanism of differential activation of the exchanger. To pursue this question, a novel antibody, anti-XB17, directed to the cytoplasmic tail of NHE1 was characterized and then utilized to examine the expression of NHE1 protein and the level of phosphorylation of the serum stimulated exchanger in human embryonic lung fibroblasts (WI-38), SV40-transformed WI-38, and nontransformed human foreskin fibroblasts (HSWP) cells. The level of mRNA expressed in these cells was also examined. Results indicate that the parental cell lines and other nontransformed fibroblasts express NHE1. Although the transformed cell lines express NHE1 mRNA in approximately similar abundance to the parental lines, they contain decreased quantity of NHE1 exchanger/mg membrane protein as recognized by anti-XB17 Ab. The mechanism that results in the apparent decrease in NHE1 protein levels in the transformed cells is not known. Also, the SV40-transformed cells express an exchanger with a higher apparent molecular weight. The WI-38 cells demonstrate phosphorylation of NHE1 in response to mitogenic stimulation. ALthough the nontransformed HSWP cells have a high level of Na+/H+ exchanger protein, they do not show a significant increase in phosphorylation following serum stimulation, when examined by immunoprecipitation, and analysis on 1-D gels. However, subsequent studies of tryptic phosphopeptides from the immunoprecipitated exchanger reveal that serum-stimulated phosphorylation of one tryptic peptide does occur but may be masked in the first dimension by differential phosphorylation of other tryptic peptides that are more heavily phosphorylated in unstimulated cells.

Selective Cytotoxicity of Transformed Cells but Not Normal Cells by a Sialoglycopeptide Growth Regulator in the Presence of Tumor Necrosis Factor

The tumor necrosis factor-α (TNF)-resistant, SV40-transformed, murine fibroblast cell lines, F5b and F5m, became sensitive to TNF-mediated cytolysis after treatment with a biologically active 18 kDa peptide fragment (SGP) derived from a 66-kDa parental cell surface sialoglycoprotein. Neither TNF nor the SGP alone exhibited cytotoxicity to the two SV40-transformed cell lines. However, Balb/c 3T3 cells, incubated with SGP alone or with SGP and TNF, were not killed. Therefore, SGP can selectively sensitize cells for TNFα-mediated cytotoxicity. This selective sensitization may be due to the previously documented ability of the SGP to selectively mediate cell cycle arrest.

Heat shock-induced cell death in murine microvascular endothelial cells depends on priming with tumor necrosis factor-alpha or interferon-gamma.

We previously reported that sequential exposure of cultured porcine aortic endothelial cells to lipopolysaccharide (LPS) and then to inducers of the heat shock response resulted in lethal injury by programmed cell death. In these present experiments, we evaluated the ability of other mediators of sepsis to prime endothelial cells for subsequent injury upon heat shock induction. We used SVEC4-10 microvascular endothelial cells, a cloned SV40-transformed cell line derived from LPS-resistant C3H/HeJ mice. When these endothelial cells were treated first with tumor necrosis factor-alpha followed by induction of the heat shock response with either heat or sodium arsenite (As), a standard chemical inducer of the heat shock response in vitro, a tumor necrosis factor dose-dependent cytotoxicity was observed, similar to that which we had seen previously in porcine endothelial cells primed with LPS. Subsequent experiments found that priming with interferon-gamma produced a similar dose-dependent toxicity upon heat shock with either heat or As. The reducing agents dithiothreitol and n-acetylcysteine, which we have previously shown to inhibit heat shock-induced programmed cell death in LPS-primed porcine endothelial cells, were also effective in protecting against heat shock-induced death following cytokine-priming in this microvascular cell line, suggesting that the intracellular signaling pathways of these priming agents are somewhere convergent. These data suggest that both exogenous and endogenous mediators of inflammation can prime endothelial cells for subsequent injury upon induction of the heat shock response, and are consistent with the emerging concept of redundancy in inflammatory signaling.

Loss of apc protein expressed by human colonic epithelial cells and the appearance of a specific low‐molecular‐weight form is associated with apoptosis IN vitro

APC (adenomatous polyposis coli) protein is differentially expressed in the normal colonic crypt and believed to be involved in colonic cell maturation. In this work we investigated whether expression of the APC protein is associated with cell death in colonic epithelial cells. We have previously reported an in vitro system to study apoptosis. Briefly, cells attached to the flask have a low frequency of apoptosis (1-3%), whereas cells that detach from the flask and float in the medium have a high proportion of apoptotic cells (36-96% depending on the cell line). The full-length 300-kDa or truncated APC protein, normally expressed by the attached cells (detected using the FE9 antibody), was found to be lost in the floating apoptotic cells in 8/11 colon tumour cell lines examined. In addition, the APC antibody FE9 detected a 90-kDa protein in the floating apoptotic cells of all cell lines investigated, which was not present in attached cells. Furthermore, loss of full-length APC and gain of the 90-kDa protein was observed in the apoptotic cells of 2 cell lines derived from other tissues: the SV40-transformed fibroblast cell line CMSV40fib and the lymphoblastoid B-cell line BJA-B. In cells repeatedly frozen and thawed, believed to induce necrotic cell death, full-length or truncated APC was also lost, though a 95-kDa protein distinct from that in apoptotic cells was observed. Specific loss of full-length or truncated APC (resulting in a 90-kDa protein in apoptotic cells but a 95-kDa protein in necrotic cells) is therefore associated with cell death. Our findings suggest a possible role for APC in cell survival.

Immunization of BALB/c mice with recombinant simian virus 40 large tumor antigen induces antibody-dependent cell-mediated cytotoxicity against simian virus 40-transformed cells. An antibody-based mechanism for tumor immunity.

Abstract In this report, we describe an immune mechanism that seems to play a role in tumor immunity in BALB/c mice challenged with SV40-transformed cells. This immune mechanism was induced by immunization of mice with rSV40 large tumor Ag (T-Ag). After rSV40 T-Ag immunization, BALB/c mice were protected from a lethal tumor challenge with syngeneic SV40-transformed cells (mKSA). Specifically, we examined CTL activity, NK activity, complement-dependent cytotoxicity, and Ab-dependent cell-mediated cytotoxicity (ADCC) in rSV40 T-Ag-immunized mice that were protected from a subsequent lethal tumor challenge. Immune splenocytes from surviving animals that were subsequently primed in vitro with rSV40 T-Ag demonstrated little to no SV40 T-Ag-specific CTL activity. In addition, natural immunity, as assessed by NK lytic activity, was comparable to that of unimmunized animals. Similarly, minimal significant complement-dependent cytotoxicity of radiolabeled mKSA cells was demonstrated by SV40 T-Ag-specific serum Abs. However, SV40 T-Ag-specific Abs using peritoneal exudate cells as effectors, exhibited significant levels of ADCC against radiolabeled mKSA target cells. Together, these studies indicate that rSV40 T-Ag immunization elicited ADCC that may represent a potential humoral immune mechanism involved in the protection of BALB/c mice from a lethal challenge with syngeneic SV40-transformed cells.

Distribution of apoptosis-mediating Fas antigen in human skin and effects of anti-Fas monoclonal antibody on human epidermal keratinocyte and squamous cell carcinoma cell lines.

Fast antigen is a cell surface protein that mediates apoptosis. Using immunohistological, flow cytometry and electron microscopic analyses, we investigated the expression of Fas antigen on various skin tissues, and on cultured SV40-transformed human epidermal keratinocyte cell line KJD and human skin squamous cell carcinoma cell line HSC. The Fas antigen was widely distributed in skin components such as the keratinocytes in the lower portion of the epidermis, epidermal dendritic cells, endothelial cells, fibroblasts, apocrine glands, eccrine sweat glands, sebaceous glands, some normal melanocytes and infiltrating lymphoid cells. It was also strongly expressed on the keratinocytes of lichenoid eruptions seen in lupus erythematosus and lichen planus, and on the spongiotic or acanthotic epidermis seen in chronic eczema, adult T-cell leukaemia/lymphoma (ATLL) and atopic dermatitis. Its expression was closely correlated with lymphoid infiltrating cells and it was strongly expressed in lymphoid neoplastic cells, particularly ATLL cells, and fibroblasts seen in dermatofibroma. However, the antigen was not detected on basal cell epithelioma cells, some malignant melanomas or any junctional naevi. The cell lines KJD and HSC strongly expressed the Fas antigen, and crosslinking of the Fas antigen by an anti-Fas monoclonal antibody induced apoptosis of these cell lines. These results indicate that the apoptosis-mediating Fas antigen may play an important role in normal skin turnover and cell differentiation, in immune regulation of skin tumours, and in the pathogenesis of various skin diseases.

Modulation of alpha-actinin levels affects cell motility and confers tumorigenicity on 3T3 cells.

alpha-Actinin is an abundant actin crosslinking protein, also localized at adherens type junctions. In adhesion plaques, alpha-actinin can link the actin filaments to integrin via vinculin and talin, or directly by binding to the cytoplasmic domain of beta 1-integrin. The expression of alpha-actinin is rapidly elevated in growth-activated quiescent cells, and is reduced in SV40-transformed 3T3 cells and various differentiating cell types (reviewed by Glück, U., Kwiatkowski, D. J. and Ben-Ze'ev, A. Proc. Nat. Acad. Sci. USA 90, 383-387, 1993). To study the effect of changes in alpha-actinin levels on cell behavior, alpha-actinin expression was elevated in 3T3 cells by transfection with a full-length human nonmuscle alpha-actinin cDNA. To suppress alpha-actinin levels, 3T3 cells were transfected with an antisense alpha-actinin cDNA construct. Cells overexpressing alpha-actinin by 40-60% displayed a significant reduction in cell motility, as demonstrated by their slower locomotion into an artificial wound, and by forming shorter phagokinetic tracks on colloidal gold-coated substrata. 3T3 cells in which the expression of alpha-actinin was reduced to 25-60% of control levels, after antisense alpha-actinin transfection, had an increased cell motility. Moreover, such alpha-actinin-deficient 3T3 cells formed tumors upon injection into nude mice. The results demonstrate that modulations in alpha-actinin expression can affect, in a major way, the motile and tumorigenic properties of cells, and support the view that decreased alpha-actinin expression could be a common regulatory pathway to malignant transformation of 3T3 cells.