Recombinant hydrophilic human gp100: uptake by dendritic cells and stimulation of autologous CD8+ lymphocytes from melanoma patients.

Native gp100, a glycoprotein highly expressed in the majority of melanomas, contains several immunogenic peptides that are recognized by cytotoxic lymphocytes (CTLs) in the context of major histocompatibility complex (MHC) class I molecules. The objective of this study was to evaluate the ability of dendritic cells (DCs) from melanoma patients to take up gp100 protein and stimulate specific autologous CTL. The gp100 used in this study was a recombinant molecule with diminished hydrophobicity, HR-gp100, produced in Escherichia coli bacteria and in Pichia pastoris yeast.

MHC class I-independent recognition of NK-activating receptor KIR2DS4.

Natural killer cells are capable of killing tumor and virus-infected cells. This killing is mediated primarily via the natural cytotoxicity receptors, including NKp46, NKp44, NKp30, and by the NKG2D receptor. Killer cell Ig-like receptors (KIRs) are mainly involved in inhibiting NK killing (inhibitory KIRs) via interaction with MHC class I molecules.

Production and purification of melanoma gp100 antigen and polyclonal antibodies.

gp100 is a melanoma-associated antigen found to carry immunogenic epitopes that can induce a CTL response against tumor cells. Production and purification of large quantities of this polypeptide may be important in the context of diagnosis and vaccinating against melanoma. To overcome the hydrophobic nature of gp100, we cloned and expressed only a part of the protein, and obtained a hydrophilic recombinant polypeptide (HR-gp100) that contained most of the immunogenic peptides.

Interleukin-2 improves tumour response to DNP-modified autologous vaccine for the treatment of metastatic malignant melanoma.

This paper is a report of response rate (RR) and survival of 34 metastatic melanoma patients who received a dinitrophenyl (DNP)-modified autologous melanoma cell vaccine. In all, 27 patients started the vaccine as a primary treatment for metastatic melanoma and seven started it as an adjuvant, with no evidence of disease at the time, but had developed new metastases. Interleukin-2 (IL-2) was administered in 24 out of the 34 patients: 19 who progressed on vaccine alone and five who had the combination from start.

Caspase-mediated cleavage converts Livin from an antiapoptotic to a proapoptotic factor: implications for drug-resistant melanoma.

Inhibitor of apoptosis protein (IAP) is a family of intracellular proteins that plays an essential role in the regulation of apoptosis. Recently, we and others discovered a new member of this family, termed Livin. Many studies have focused on the inhibitory effect of IAPs on caspases.

Cytogenetic analysis of melanoma cell lines: subclone selection in long-term melanoma cell cultures.

We employed G-banding cytogenetic analysis to follow the clonal constitution of short-term cultures of metastatic malignant melanoma compared to their long-term cultures. Eight metastatic melanoma cell lines were analyzed. No long-term culture was found to be identical to its line of origin.

Autologous cell vaccine as a post operative adjuvant treatment for high-risk melanoma patients (AJCC stages III and IV). The new American Joint Committee on Cancer.

This study evaluates the overall survival and disease free survival of melanoma patients that were treated with an autologous melanoma cell vaccine, administered as a post-operative adjuvant. Included are 43 patients with totally resected metastatic melanoma (28-AJCC stage III, 15-AJCC stage IV), with a median follow up of 34 months (6-62). The treatment consisted of eight doses of a vaccine made of 10-25x10(6) autologous melanoma cells either released from the surgical specimen or grown in cell cultures.

Synergistic inhibitory effects of genistein and tamoxifen on human dysplastic and malignant epithelial breast cells in vitro.

Objective: Genistein is a phytoestrogen with in vitro anticancerogenic activity. We examined in vitro the effects of genistein alone, or in combination with estradiol and tamoxifen, on the growth of human dysplastic and malignant epithelial breast cell lines.

Methods: Dysplastic breast cell lines (MCF-10A(1), MCF-ANeoT, MCF-T(6)3B) and cell lines of breast cancer (MCF-7, MDA-231, MDA-435) were cultured as monolayers in RPMI 1640 medium supplemented with 10% fetal bovine serum, and L-glutamine.

CD66a interactions between human melanoma and NK cells: a novel class I MHC-independent inhibitory mechanism of cytotoxicity.

NK cells are able to kill virus-infected and tumor cells via a panel of lysis receptors. Cells expressing class I MHC proteins are protected from lysis primarily due to the interactions of several families of NK receptors with both classical and nonclassical class I MHC proteins. In this study we show that a class I MHC-deficient melanoma cell line (1106mel) is stained with several Ig-fused lysis receptors, suggesting the expression of the appropriate lysis ligands.

Interleukin-6 secretion in mice is associated with reduced glucose-6-phosphatase and liver glycogen levels.

Mice bearing interleukin-6 (IL-6)-secreting tumor were used to study the chronic effect of IL-6 on carbohydrate metabolism. Mice were injected with allogeneic tumor cells transduced with the murine IL-6 gene. Serum IL-6 levels were correlated exponentially with tumor weight.

Tumor necrosis factor inhibits the transcriptional rate of glucose-6-phosphatase in vivo and in vitro.

Recombinant human tumor necrosis factor-alpha (TNF) injection in mice was associated with a reduced blood glucose level, already manifest 6 hours following cytokine administration. Insulin levels were not affected. Glycogen content was decreased in a dose-dependent and time-response manner.

[Antimutagenic properties of substances containing beta-carotene].

Two carotene-containing drugs (natural and artificial) were shown to decrease the rate of chromosomal aberrations, induced by cyclophosphane, in bone marrow cells of mice, daily ration of which contained 10-20 mg/kg of beta-carotene within 1-3 weeks. The rate of chromosomal aberrations induced was decreased 1.5-2-fold in various experiments.

[Relation between DNA replication and neutral Mn 2+-dependent DNAse activity in chromatin].

In cultures of primary murine fibroblasts the 10% serum stimulates the replicative synthesis of DNA inhibited by aphidicolin and araC (cytosine arabinoside). Using direct immunofluorescence analysis, it was shown that antibodies penetrate inside the cells and after 4 hours are pooled in the nuclei, where they remain for another 20 hours. The substitution of antibodies against chromatin DNAase by bovine serum albumin of normal serum gamma-globulins does not interfere with the DNA synthesis induction.

[Ability of virus SV40 T-antigen to simulate the effect of specific T lymphocyte growth factor--interleukin-2].

The entering of T-lymphocytes into the DNA-synthesizing phase was marked by three consecutive signals, i.e., antigenic influence, interleukin-2, a specific T-lymphocyte cell growth factor, and non-specific serum growth-promoting factors, in the first place, transferrin.

[Induction of chromosome aberrations by the T antigen of the SV40 virus introduced into the cells using liposomes].

A purified SV40 T antigen introduced into hamster cells by means of liposomes accumulated in the nuclei within 10 h and persisted there further as long as 10 to 12 h. Within the first day after cell treatment with T antigen numerous chromosome aberrations including breaks, translocations and gaps were observed in the cells. The number of aberrations slightly reduced by the 2nd day followed by restoration of the normal cell karyotype by the 5th day.

[Possible role of the T antigen in inducing chromosome aberrations in SV40 virus-transformed cells].

A possible role of the simian virus 40 T antigen in chromosome damages in transformed cells was examined. Two lines of Golden hamster embryonal fibroblasts, transformed by SV40 tsA30 and ts239 mutants (He30 and He239, respectively), were incubated at nonpermissive (40.5-41 degrees C) or permissive (33 degrees C) temperatures.

[Induction of the replicative synthesis of DNA by SV40 virus T antigen introduced into cells using liposomes].

The role of virus SV40 T-antigen in the induction of cell DNA synthesis during its incorporation into cell liposomes was studied, using monolamellar liposomes obtained by phase reversal with incorporated highly purified T-antigen. Immunofluorescence studies revealed that T-antigen effectively penetrates inside the cells and after 10 hours is accumulated in the nuclei, where its level remains unchanged for 24 hours. Injections of purified T-antigen into the renal cells of serum-starved CV1 monkeys resulted in an almost 10-fold increase in the number of DNA-synthesizing cells 18 hours after the exposure.

[Interaction of SV40 T-antigen with tumor cell chromatin].

The T-antigen of SV40 virus can be found in purified chromatin prepared from virus-induced tumour cells of the Syrian hamster. After treatment of chromatin or isolated nuclei with micrococcal nuclease this protein is detected in the high molecular weight and oligonucleosomal fractions. Data from sedimentation analysis and gel electrophoresis suggest that the T-antigen is predominantly linked with the oligonucleosomal fraction and in a lesser degree with mononucleasomes containing linker DNA and histone H1.

[Interaction of SV40 T-antigen with homologous and heterologous DNA].

Using the DNA filter binding assay, the effects of ionic strength and pH on SV40 T-antigen interaction with viral DNA were studied. The apparent association constants for T-antigen binding to SV40 DNA in Scatchard coordinates in the presence of 40 mM NaCl are equal to 0.67 .

[Existence of a form of SV40 T antigen incapable of interacting with DNA].

The interaction of SV40 T-antigen and viral DNA was studied by using adsorption of DNA-protein complexes on nitrocellulose filters. The T-antigen purification procedure included ion-exchange chromatography on DEAE-cellulose, selective adsorption of cellular proteins on single-stranded DNA-cellulose, chromatography on heparin-Sepharose and removal of cell proteins by an immunosorbent. Only the latter step allowed to remove the contamination of cellular DNA-binding proteins, judging from the reaction of T-antigen neutralization by specific antibodies.

[Possible role of SV40 T antigen in cell transformation].

The effect of purified SV40 T antigen on DNA synthesis in isolated nuclei from the confluent culture of CV-1 cells was studied. In the presence of T antigen the incorporation of [3H]TTP into DNA was found to be 2 to 3 times as high as in the control nuclei. The resulting labelled DNA was subjected to alkaline sucrose gradient centrifugation, which revealed the presence of 4S DNA species, corresponding to Okazaki fragments of animal cells.

Somatic hybridization and oncogenesis; (Mechanism of formation of malignant tumors and metastases by the action of antilymphocytic serum).

The results of experiments carried out to test some of the consequences of the earlier general theory of oncogenesis, according to which the malignant tumor cell can arise as a result of somatic hybridization of cells of different organ- and tissue-specificity, are described. In the first series a tumor induced by cellophane film, was grafted into syngeneic and allogeneic mice, and antilymphocytic serum (ALS) was then injected. Metastases occurred only in allogeneic recipients receiving ALS.

[The potential nuclease activity of the T antigen of SV-40 virus].

SV40 T-antigens were isolated from an extract of golden hamster tumours by precipitation with ammonium sulphate with subsequent fractionation on DEAE cellulose. The degree of purification of the preparation proved to be about 100-fold; it, however, contained an admixture of several cell proteins. Treatment of the DNA of the calf thymus with the T-antigen preparation in the presence of magnesium ions decreased the viscosity of the DNA solution during the first hour of incubation.