Biomedical subjects

E Gorelik

Publications and source records attributed to E Gorelik.

At least 19 recordsLinked to original sources

PubMed2005

Multianalyte profiling of serum cytokines for detection of pancreatic cancer.

Early detection of pancreatic cancer might improve clinical outcome. Significant alterations in the levels of individual serum cytokines have been reported in pancreatic cancer. We hypothesized that a multicytokine panel could serve as biomarkers for pancreatic cancer. To evaluate the diagnostic utility of such a panel, we have utilized a novel multianalyte LabMAP profiling technology that allows simultaneous measurement of multiple markers. In this study, a panel of 31 serological markers including cytokines, chemokines, growth and angiogenic factors in combination with CA 19-9 was analyzed in sera of pancreatic cancer patients, patients with chronic pancreatitis, and matched control healthy subjects. Statistical analysis identified a multicytokine panel that was able to distinguish pancreatic cancer from healthy controls with a sensitivity of 85.7% and specificity of 92.3%, which was superior to performance of CA 19-9 alone. Importantly, a multicytokine panel allowed the discrimination of pancreatic cancer from chronic pancreatitis with high sensitivity of 98% and specificity of 96.4%. In conclusion, we demonstrated that analysis of multiple serum cytokines using a novel LabMAP technology is a promising approach for development of a diagnostic assay for pancreatic cancer.

Biomarkers, Tumor↗

PubMed2001

Anoikis and metastatic potential of cloudman S91 melanoma cells.

Anoikis is a form of apoptosis induced in normal cells as a result of loss of their adhesion to substrate. In the present study, we have tested whether tumor cells are also sensitive to anoikis and whether selection of tumor cells for resistance to anoikis could increase their metastatic ability. In vitro cultured Cloudman S91 melanoma cells are strongly adherent to the plastic. Prevention of their adherence by rocking or by covering culture plates with polyhydroxyethylmethacrylate resulted in induction of anoikis and death of almost all cells. Their death was prevented in the presence of caspase inhibitor Z-Val-Ala-Asp-fluoromethyl ketone. To select anoikis-resistant cells, S91 cells floating in the culture medium were sequentially isolated and transferred for seven generations. As a result, a new subline of S91 cells capable of growing in free cell suspension was selected. These S91 nonadherent (S91Nadh) cells were completely resistant to anoikis and manifested higher metastatic ability than S91Adh cells. Anoikis resistance of S91Nadh cells was not attributable to their resistance to other apoptotic signals in vitro, and they showed no increase in their survival in vivo in the lungs after i.v. inoculation. Increased metastatic potential of the anoikis-resistant S91Nadh cells was associated with various phenotypic changes, including increased proliferation and loss of VLA-4 integrin expression because of down-regulation of the VLA-49alpha (CLD49d) gene. In parallel, they showed a reduction in homotypic aggregation and binding to endothelial cells, increased Matrigel invasiveness, and decreased matrix metalloproteinase-2 and matrix metalloproteinase-9 activity that paralleled up-regulation of the TIMP-1 gene. S91Nadh cells also manifested changes in cell surface carbohydrates, such as appearance of alpha-galactosyl epitopes as a result of up-regulation of the alpha1,3-galactosyltransferase gene and concomitant reduction in cell membrane sialylation. Thus, selection of S91 melanoma cells for anoikis resistance resulted in an increase in their metastatic potential in parallel with multiple alterations in their phenotypic properties.

Animals↗

PubMed2001

Soluble recombinant endostatin purified from Escherichia coli: antiangiogenic activity and antitumor effect.

Endostatin is a potent and specific antiangiogenic protein capable of inhibiting the growth of murine and xenotransplanted human tumors. Thus far, however, recombinant endostatin prepared from Escherichia coli is insoluble after purification and therefore inappropriate for clinical settings. A soluble form of endostatin is available from a yeast system with relatively low yield and high cost, which has made it difficult to produce endostatin in quantities sufficient for extensive clinical evaluation. In this study, we developed a protocol to generate soluble recombinant murine endostatin from E. coli at a yield of 150 mg/liter-culture and 99% purity. The in vivo antiangiogenic and antitumor activities of the soluble recombinant endostatin are equally as potent as those of the previously published insoluble form. A similar protocol may be used to produce soluble human endostatin.

Angiogenesis Inhibitors↗

PubMed2001

On the role of cell surface carbohydrates and their binding proteins (lectins) in tumor metastasis.

This review focuses on the recent advances in investigations of the role of cell surface carbohydrates in tumor metastasis. It also summarizes the results of extensive studies of endogenous lectins, their structure, carbohydrate specificity and biological functions with the major emphasis on the significance of lectin-cell surface carbohydrate interactions in a metastatic process. Numerous data demonstrate that malignant transformation is associated with various and complex alterations in the glycosylation process. Some of these changes might provide a selective advantage for tumor cells during their progression to more invasive and metastatic phenotype. Cell glycosylation depends on the expression and function of various glycosyltransferases and glycosidases. Recently, transfection of genes encoding various glysosyltransferases gene in sense and antisense orientation helped to bring direct evidence that changes in cell surface carbohydrates are important for the metastatic behavior of tumor cells. Cell surface carbohydrates affect tumor cell interactions with normal cells or with the extracellular matrix during metastatic spread and growth. These interactions can be mediated via tumor cell carbohydrates and their binding proteins known as endogenous lectins. The family of the discovered endogenous lectins is rapidly expanding. The number of C-type lectins has reached 50 and at least 10 galectins have been identified. The biological significance of the endogenous lectins and their possible role in tumor growth and metastasis formation has started to unravel. Some lectins recognize the 'foreign' patterns of cell surface carbohydrates expressed by microorganisms and tumor cells, and play a role in innate and adaptive immunity. It was shown that lectins affect tumor cell survival, adhesion to the endothelium or extracellular matrix, as well as tumor vascularization and other processes that are crucial for metastatic spread and growth.

Animals↗

PubMed2000

Nitric oxide (NO), methylation and TIMP-1 expression in BL6 melanoma cells transfected with MHC class I genes.

We have previously found that transfection of BL6-8 melanoma cells with the H-2K, but not H-2D/L genes resulted in loss of their metastatic ability that was associated with decrease in their invasiveness and up-regulation of TIMP-1 expression. In the present study using the methylation-specific PCR (MSP) we found that lack of TIMP-1 expression in BL6-8 is associated with methylation in the TIMP-1 5' regulatory area. In the H-2Kb transfected CL8-1 melanoma cells up-regulation of TIMP-1 was in parallel with loss of TIMP-1 gene methylation. Treatment of BL6-8 with 5-azacytidine or with an inhibitor of histone deacetylase trichostatin A resulted in up-regulation of TIMP-1 expression. These results indicate that methylation and histone deacetylation play an important role in transcription repression of TIMP-1 in BL6 melanoma cells. Some data showed that nitric oxide (NO) could affect methylation and expression of various gene. Therefore we analyzed NO production in B16 melanoma cell lines with different expression of TIMP-1. We have found that B16F10 and BL6-8 melanoma cells do not express TIMP-1 and do not produce nitric oxide (NO) even after stimulation with IFN-gamma and LPS. However, BL6-8 cells transfected with H-2Kb or H-2Kd, but not H-2Dd or H-2Ld gene expressed TIMP-1 and produced NO constitutevely. NO production in these cells was further stimulated by IFN-gamma and LPS. Northern blot analysis showed that expression of iNOS was paralleled with TIMP-1 expression in the tested melanoma cells. However, NO produced by SNAP or inhibition of NO production by NMA did not affect TIMP-1 expression in the tested melanoma cells. Thus, TIMP-1 expression and NO production in BL6 melanoma cells transfected with MHC class I gene coincides but it remains unclear whether NO is responsible for the change in TIMP-1 methylation and expression.

Antimetabolites, Antineoplastic↗

PubMed1999

Loss of retrovirus production in JB/RH melanoma cells transfected with H-2Kb and TAP-1 genes.

JB/RH1 melanoma cells, as well as other melanomas of C57BL/6 mice (B16 and JB/MS), express a common melanoma-associated antigen (MAA) encoded by an ecotropic melanoma-associated retrovirus (MelARV). JB/RH1 cells do not express the H-2Kb molecules due to down-regulation of the H-2Kb and TAP-1 genes. When JB/RH1 cells were transfected with the H-2Kb and cotransfected with the TAP-1 gene, it resulted in the appearance of H-2Kb molecules and an increase in their immunogenicity, albeit they lost expression of retrovirus-encoded MAA recognized by MM2-9B6 mAb. Loss of MAA was found to result from a complete and stable elimination of ecotropic MelARV production in the H-2Kb/TAP-1-transfected JB/RH1 cells. Northern blot analysis showed no differences in ecotropic retroviral messages in MelARV-producing and -nonproducing melanoma cells, suggesting that loss of MelARV production was not due to down-regulation of MelARV transcription. Southern blot analysis revealed several rearrangements in the proviral DNA of H-2Kb-positive JB/RH1 melanoma cells. Sequence analysis of the ecotropic proviral DNA from these cells showed numerous nucleotide substitutions, some of which resulted in the appearance of a novel intraviral PstI restriction site and the loss of a HindIII restriction site in the pol region. PCR amplification of the proviral DNAs indicates that an ecotropic provirus found in the H-2Kb-positive cells is novel and does not preexist in the parental H-2Kb-negative melanoma cells. Conversely, the ecotropic provirus of the parental JB/RH1 cells was not amplifable from the H-2Kb-positive cells. Our data indicate that stable loss of retroviral production in the H-2Kb/TAP-1-transfected melanoma cells is probably due to the induction of recombination between a productive ecotropic MelARV and a defective nonecotropic provirus leading to the generation of a defective ecotropic provirus and the loss of MelARV production and expression of the retrovirus-encoded MAA.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

Sequence and insertion sites of murine melanoma-associated retrovirus.

We previously showed that B16 melanoma cells produce ecotropic melanoma-associated retrovirus (MelARV) which encodes a melanoma-associated antigen recognized by MM2-9B6 monoclonal antibody. The biological significance of MelARV in melanoma formation remains unknown. We found that infection of normal melanocytes with MelARV resulted in malignant transformation. It is likely that MelARV emerged from the defective Emv-2 provirus, a single copy of ecotropic provirus existing in the genome of C57BL/6 mice. In the present study, we cloned and sequenced the full-length MelARV genome and its insertion sites and we completed sequencing of the Emv-2 provirus. Our data show that MelARV has a typical full-length retroviral genome with high homology (98.54%) to Emv-2, indicating a close relationship between both viruses. MelARV probably emerged as a result of recombination between Emv-2 and an endogenous nonecotropic provirus. Some observed differences in the gag and pol regions of MelARV might account for the restoration of productivity and infectivity of a novel retrovirus that somatically emerged during melanoma formation. MelARV does not contain any oncogene and therefore might induce transformation by insertional mutagenesis. We sequenced two insertion sites of MelARV. The first insertion site represents the 3' coding region of the c-maf proto-oncogene at 67.0 centimorgans (cM) on chromosome 8. The c-maf proto-oncogene encodes a basic leucine zipper protein homologous to c-fos and c-jun. Insertion of MelARV in BL6 melanoma cells resulted in the up-regulation of c-maf. It is noteworthy that the Emv-2 provirus is also inserted into a noncoding region at 61.0 cM on the same chromosome 8. The second insertion site is the 3' noncoding region of the DNA polymerase gamma (PolG) gene on chromosome 7. The expression of PolG was not affected by the MelARV insertion. Further investigation of the biological significance of MelARV in melanoma formation is being undertaken.

Animals↗

PubMed1998

Ecotropic C-type retrovirus of B16 melanoma and malignant transformation of normal melanocytes.

We reported previously that B16,JB/RH and JB/MS melanomas of C57BL/6 mice express the common melanoma-associated antigen (MAA) recognized by MM2-9B6 monoclonal antibody (MAb). This MAA is encoded by the env gene of an ecotropic MuLV-type retrovirus that somatically emerged in melanomas of C57BL/6 mice. The potential role of this melanoma-associated retrovirus (MelARV) in melanoma formation remains unknown and has not been previously investigated. To test this, normal melanocyte lines (melan-a and C57M) of C57BL/6 mice were infected with the MelARV produced by B16BL6 melanoma. Infection of these melanocytes with the MelARV was associated with the appearance of the MAA recognized by MM2-9B6 MAb. Most of the infected melanocyte sublines were able to grow only in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA). Two infected melanocyte sublines showed morphological changes, were able to grow in the absence of TPA and, after inoculation into C57BL/6 mice, produced rapidly growing, highly pigmented tumors. These new melanomas, derived from the MelARV-infected melan-a and C57M melanocytes, were termed Meli-A1 and Meli-BL, respectively. Southern blot analysis of EcoRI- and HindIII-digested DNAs from these melanomas showed several retroviral insertion sites. One copy of MeIARV was found to be inserted at the end of the 6th leucine domain of the c-maf proto-oncogene, which encodes a basic region/leucine zipper transcription factor related to the AP-1 family that is able to form homodimers or heterodimers with Fos and Jun transcription factors. Our data indicate that c-maf is a common insertion site of MelARV in BL6, Meli-A1 and Meli-BL melanomas, whereas no such insertion site was found in the melanocytes infected with MelARV but not malignantly transformed. Thus, our data imply that the ecotropic MelARV that somatically emerged in B16 and other melanomas of C57BL/6 mice may play a role in malignant transformation.

Animals↗

PubMed1998

Inhibition of VLA-4 and up-regulation of TIMP-1 expression in B16BL6 melanoma cells transfected with MHC class I genes.

The effect of MHC class I gene transfection on the metastatic properties of B16BL6 melanoma cells was investigated. BL6-8 melanoma cells transfected with H-2Kb or H-2Kd, but not H-2Dd or H-2Ld, genes showed a dramatic reduction in their ability to generate experimental metastases in immunosuppressed CB6F1 mice. This observation suggested that some changes in the metastatic phenotype may have been induced in the H-2K- transfected melanoma cells. Analyses of adhesive and invasive properties of BL6-8 melanoma cells transfected with H-2 class I genes have been performed. We found that the loss of metastatic properties in the H-2Kb or H-2Kd gene-transfected melanoma cells was associated with reduced adherence to endothelial cells, laminin and collagen IV, decreased ability to form homotypic cell aggregates and with a complete loss of VLA-4 integrin expression. In addition, BL6-8 melanoma cells transfected with H-2K genes demonstrated reduced ability to invade Matrigel that paralleled up-regulation of TIMP-1 expression. Incubation of untransfected BL6-8 clone or B16F1 cells with 5-azacytidine similarly resulted in up-regulation of TIMP-1, suggesting that the changes in methylation of TIMP-1 gene could be responsible for TIMP-1 expression in the H-2K-transfected BL6-8 melanoma cells. Transfection of BL6-8 cells with the H-2Dd/Ld genes did not affect their adhesive and invasive properties. Previously we reported that reduction in the metastatic properties of the H-2Kb transfected cells was associated with alterations in cell surface carbohydrates with appearance of alpha-galactosyl epitopes and reduction in cell surface sialylation. The present data indicate that, in addition to changes in cell surface carbohydrates, reduction in adhesive properties and up-regulation of TIMP-1 may be responsible for the observed loss of metastatic potential of BL6-8 cells transfected with the H-2K genes.

Animals↗

PubMed1997

Reduction of metastatic properties of BL6 melanoma cells expressing terminal fucose(alpha)1-2-galactose after alpha1,2-fucosyltransferase cDNA transfection.

We reported previously that transfection of BL6 melanoma cells that do not express the alpha1,3-galactosyltransferase (alpha1,3GT) gene with the alpha1,3GT cDNA resulted in synthesis and expression of alpha-galactosyl epitopes (Gal(alpha)1-3Gal(beta)1-4GlcNAc-R) and an impairment of their metastatic potentials. It was of interest to test whether inhibition of metastatic properties of BL6 melanoma cells is specifically associated with the appearance of the terminal alpha-Gal or whether capping N-acetyllactosamine with another oligosaccharide would also affect the metastatic properties of BL6 melanoma cells. For this purpose, BL6-2 clone isolated from B16BL6 melanoma was transfected with the alpha1,2-fucosyltransferase (alpha1,2FT) cDNA. The alpha1,2FT catalyzes a transglycosylation reaction, resulting in syntheses of the Fuc(alpha)1-2Gal(beta)1-4GlcNAc-R structure, which is known as the H antigen of O blood group in humans and is also synthesized in some cells of mice. Transfection of BL6 melanoma cells with the alpha1,2FT cDNA resulted in the appearance of the terminal Fuc(alpha)1-2Gal(beta)1-4GlcNAc-R epitopes reacting with the Ulex europaeus agglutinin lectin. In parallel, the transfected cells showed a decrease in N-acetyllactosamine sialylation. Decline in sialylation of the transfected cells is likely to be the result of competition between alphal,2FT and alpha2,3- or alpha2,6-sialyltransferases for the common substrate N-acetyllactosamine (Gal(beta)1-4GlcNAc-R) on N-linked carbohydrate chains of glycoproteins and glycolipids. The alpha1,2FT-transfected BL6-2 cells showed an increase in homotypic aggregation. In parallel, metastatic ability of the alpha1,2FT-transfected BL6-2 cells was reduced significantly in the immunocompetent as well as immunosuppressed (X-irradiated) mice. Thus, these data imply that capping N-acetyllactosamine with alphaGal or alphaFuc and the corresponding reduction in sialylation of BL6-2 melanoma cells were associated with reduction of their metastatic potential.

Animals↗

PubMed1996

Inhibition of melanoma-associated antigen expression and ecotropic retrovirus production in B16BL6 melanoma cells transfected with major histocompatibility complex class I genes.

We have previously reported that expression of the melanoma-associated antigen (MAA) recognized by MM2-9B6 monoclonal antibody in B16 melanoma was closely associated with C-type ecotropic retroviral particle production. Our present data show that this MAA is encoded by the env gene of an ecotropic retrovirus produced by B16 melanoma cells. Transfection of H-2Kb or H-2Kd genes into two individual clones isolated from B16BL6 melanoma, BL6-8 (H-2Kb-, H-2Db+) and BL6-2 (H-2Kb-, H-2Db-), resulted in a loss of MAA expression. Electron immunohistochemical analysis of melanoma cells and reverse transcriptase assay revealed that the loss of MAA expression in the H-2K gene-transfected cells paralleled the elimination of retroviral particles. In contrast, expression of the endogenous H-2Db gene or transfection with the H-2Dd or H-2Ld gene had no effect on MAA expression or retrovirus production. Northern blot analysis showed equivalent retroviral messages in retrovirus-producing and -nonproducing BL6 melanoma clones. Southern blot analysis revealed that H-2Kb-negative BL6 melanoma cells contain at least four different ecotropic retroviruses with different insertion sites that somatically emerged during malignant transformation or progression. Restriction enzyme analysis showed various changes in proviral DNAs from the H-2Kb- and H2Kd-transfected cells that failed to produce retroviral particles. The observed alterations in the patterns of PstI- and HindIII-digested proviral DNA were found to be due to the appearance of PstI and loss of HindIII restriction sites in the pol region as a result of several nucleotide substitutions. Thus, BL6 melanoma cells produce melanoma-specific ecotropic murine leukemia viruses that encode serologically detectable cell surface MAA. The transfection of BL6 melanoma cells with H-2Kb or H2Kd genes but not H-2Dd or H-2Ld genes resulted in a loss of MAA expression that was attributed to the changes in proviral DNA and loss of melanoma-specific ecotropic retrovirus particle production.

Animals↗

PubMed1996

Phenotypic changes and loss of melanoma-specific endogenous C-type retroviruses in BL6 melanoma cells transfected with the H-2Kb gene.

The murine B16 melanoma and its sublines are low or totally deficient in expression of the H-2Kb class I major histocompatibility complex (MHC) gene. In clones derived from the B16F10BL6 subline, expression of the transfected or endogenous H-2Kb gene resulted in alterations of various phenotypic properties of these melanoma cells, among which was the loss of melanoma-associated antigen (MAA) expression. Because our previous immunoelectron microscopy studies showed that MAA was associated with a C-type ecotropic retrovirus specific for melanomas of C57BL/6 origin, we examined the effect of class I H-2Kb as well as class II H-2IAk gene expression on retrovirus production in subclones of BL6 melanoma cells. Here we have shown that expression of the transfected or endogenous H-2Kb gene resulted in the loss of production of budding, MAA-specific, C-type retrovirus particles. Northern blot analysis demonstrated expression of ecotropic retroviral mRNAs in both particle-producing and non-producing BL6 melanoma clones. Southern blot analysis of high-molecular-weight cellular DNAs using an ecotropic env-specific DNA probe indicated that the parental BL6-8 melanoma cells contained the C57BL/6 endogenous ecotropic MuLV (Emv-2) and at least three additional, novel ecotropic retroviral DNAs. Restriction enzyme analysis of the proviral DNA suggests that the loss of retrovirus production in the H-2Kb-expressing melanoma clones is the result of multiple rearrangements in the novel proviral DNAs. Thus, phenotypic changes observed in the H-2Kb gene-transfected BL6 melanoma cells were associated with loss of endogenous melanoma-specific ecotropic retrovirus production.

Animals↗

PubMed1996

Loss of intracisternal A-type retroviral particles in BL6 melanoma cells transfected with MHC class I genes.

Electron microscopy of B16 melanoma and its sublines revealed that these cells produce numerous intracisternal A-type retroviral particles (IAPs). To identify and sequence the melanoma-associated IAPs of C57BL/6 mice, a cDNA library was constructed from IAP-producing BL6.8 cell RNA and screened using MIA14 IAP DNA as a probe. A 6-8 kb mRNA was identified that represents the full-length message for a new subfamily of IAP, termed MeIAP. The melanoma-derived IAP cDNA showed high similarity to MIA14 with major differences in the LTR. A nine base motif of the R region showed that this IAP differs from other previously sequenced IAPs. Analysis of the individual clones from BL6 melanoma revealed that IAPs were produced only in the clones that failed to express H-2Kb molecules. No IAPs were found in the melanoma clones that expressed endogenous H-2Kb. To analyse further the association between MHC class I genes and IAP production, the H-2Kb-negative clones of BL6 melanoma were transfected with various H-2 genes. Transfection of the H-2Kb or H-2Kd, but not H-2Dd or H-2Ld genes resulted in the elimination of IAPs. Northern blot analysis revealed that loss of IAPs in the H-2K gene-transfected BL6 melanoma cells was due to lack of IAP transcripts. Elimination of IAPs in the H-2Kb-positive BL6 melanoma cells was also accompanied by alterations in expression of various cellular genes and changes of their phenotypic properties.

Animals↗

PubMed1995

Alterations of cell surface carbohydrates and inhibition of metastatic property of murine melanomas by alpha 1,3 galactosyltransferase gene transfection.

Three melanomas of C57BL/6 mice (BL6, JB/MS, and JB/RH) share several phenotypic properties. All these cells contain melanoma-specific ecotropic C-type retrovirus that encodes melanoma-associated antigen recognizable by MM2-9B6 mAb. They do not express H-2Kb molecules, and the alpha-galactosyl epitopes (Gal alpha 1-3Gal beta 1-4GlcNAc-R) they fail to react with soybean agglutinin (SBA), peanut agglutinin (PNA), and vicia villosa (VV) lectins. Previously, we found that failure of BL6 melanoma cells to express alpha-galactosyl epitopes is due to down-regulation of alpha 1,3 galactosyltransferase (alpha 1,3GT) gene expression. To evaluate the possible role of alpha-galactosyl cell membrane carbohydrates in regulation of metastatic properties, individual clones isolated from BL6, JB/MS, and JB/RH melanomas were transfected with alpha 1,3GT cDNA. This resulted in appearance of alpha-galactosyl epitopes, as well as of carbohydrates reacting with SBA, PNA, or VV lectins, but did not affect expression of H-2 class I molecules or melanoma-associated antigen. Appearance of SBA, PNA, and VV lectin binding carbohydrates in the alpha 1,3GT gene-transfected melanoma cells is a result of reduction of cell membrane sialylation and unmasking of these carbohydrates. Reduction in cell membrane sialylation in the alpha 1,3GT gene-transfected melanoma cells is probably due to the competition between alpha 1,3GT with alpha 2,3 sialyltransferase or alha 2,6 sialyltransferase for the common acceptor N-acetyllactosamine in the Golgi apparatus. As a result of this competition, cell membranes of alpha 1,3GT gene-transfected melanoma cells became galactosylated and less sialylated. In parallel with alteration of cell membrane carbohydrates, transfection of the alpha 1,3GT gene leads to the loss of metastatic properties of the transfected melanoma cells in the immunocompetent and immunosuppressed C57BL/6 mice. Thus, the use of specific glycosyltransferase cDNA transfection presents direct experimental confirmation of the importance of cell membrane carbohydrates in the regulation of metastatic properties of tumor cells.

Animals↗

PubMed1995

Augmentation of TNF cytotoxicity by protein kinase C inhibitors: role of arachidonic acid and manganese superoxide dismutase.

The protein kinase C (PKC) inhibitors bisindolylmaleimide, calphostin C, H-7 and staurosporine were examined for their effect on tumour necrosis factor (TNF) cytotoxic activity. PKC inhibitors potentiated the cytotoxic activity of TNF in TNF-sensitive cell lines (CL8-1 melanoma and WEHI-164 fibrosarcoma), but had no effect on TNF cytolytic activity in TNF-resistant cells (BL6-8 melanoma and MCA-102 fibrosarcoma). The mechanism(s) of PKC inhibitor-mediated potentiation of the cytotoxic activity of TNF was investigated by analysing the effect of PKC inhibitors on TNF-induced arachidonic acid release in TNF-sensitive and -resistant cells. TNF induced the release of arachidonic acid in TNF-sensitive cells but had no effect in TNF-resistant cells. The combination of PKC inhibitor and TNF potentiated the release of arachidonic acid from TNF-sensitive cells, but failed to induce arachidonic acid release in TNF-resistant cells. Kinetic analysis of TNF-induced arachidonic acid release in CL8-1 melanoma cells revealed that it was an early event which preceded TNF tumour lytic activity. TNF was further shown to induce manganese superoxide dismutase (MnSOD) production in TNF-sensitive cells but failed to induce MnSOD activity in TNF-resistant BL6-8 and MCA 102 cells. MnSOD acts as a scaveneger of toxic superoxide radicals and its induction by TNF paralleled arachidonic acid release. Although the PKC-selective inhibitor bisindolylmaleimide potentiated TNF-induced release of arachidonic acid, it blocked TNF-mediated induction of MnSOD in CL8-1 melanoma and WEHI-164 fibrosarcoma cells.(ABSTRACT TRUNCATED AT 250 WORDS)

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1995

Impairment of the melanogenic pathway in B16 melanoma cells transfected with class I H-2 genes.

Transfection of class I H-2Kb or H-2Kd into cells of a pigmented subclone of B16-F10 BL6 (termed BL6-8) results in the loss of melanin production. In contrast, transfected BL6-8 cells expressing H-2Dd, H-2Ld, class I H-21Ak and/or the neor genes maintained their pigmented phenotype. Melanogenesis was also inhibited in cells which expressed the endogenous H-2Kb, but not the endogenous H-2Db, gene. In order to identify the specific defects in the melanogenic pathway responsible for the absence of melanin production, factors known to be related to the regulation of pigment formation were evaluated in H-2K-expressing cells. These studies showed that: (1) transfection of BL6-8 cells with the H-2Kb or H-2Kd, but not with the H-2Dd, H-2Ld or H-21Ak, genes was associated with complete inhibition of tyrosinase activity; (2) alpha-melanocyte-stimulating hormone (MSH) and theophylline (an inhibitor of cAMP phosphodiesterase) failed to stimulate tyrosinase activity in H-2K-positive cells, whereas tyrosinase activities in untransfected, or H-2DdH-2Ld, neor or H-21Ak-transfected cells were dramatically increased by those agents; (3) treatment with MSH had no effect on cAMP levels in H-2K-positive cells but stimulated cAMP levels more than 100-fold in H-2K-negative cells; (4) in contrast to MSH, forskolin, a stimulator of adenylate cyclase, was able to stimulate cAMP levels in all cell lines tested, but in H-2Kb-positive cells the levels of forskolin-induced cAMP were significantly less than those elicited in H-2Kb-negative cells; (5) electron microscopy showed that H-2K-positive cells lacked mature melanosomes; (6) Northern blot analyses showed that H-2K-positive cells lacked mRNA for tyrosinase or for the MSH receptor. Taken together, expression of the endogenous or transfected H-2K gene in BL6 melanoma cells results in down-regulation of the entire melanogenic pathway, including the inhibition of tyrosinase and MSH receptor gene expression, cAMP responses and melanosomal biogenesis.

Animals↗

PubMed1994

Partial purification of murine tumor-associated peptide epitopes common to histologically distinct tumors, melanoma and sarcoma, that are presented by H-2Kb molecules and recognized by CD8+ tumor-infiltrating lymphocytes.

The B16/BL6 melanoma is a relatively nonimmunogenic tumor expressing low levels of MHC class I molecules. BL6 clones expressing transfected H-2Kb class I molecules were, by contrast, highly immunogenic in immunocompetent mice. Tumor-infiltrating lymphocytes (TILs) generated from the H-2Kb+ BL6 lesions (Thy 1.2+, CD8+, CD4-) efficiently lysed H-2Kb+ melanoma (CL8-1 and 2Kb38) and the H-2Kb+ nonrelated 3-methylcholanthrene (MCA)-105 sarcoma, but not the H-2Kb- parental melanoma BL6-8. This strongly suggests that CL8-1, 2Kb38, and MCA-105 express identical or cross-reactive T cell epitopes recognized by CL8-1 TILs in the context of the H-2Kb class I allele. To identify the T cell epitopes, peptides were acid-eluted from various cells, and fractionated by HPLC. Five HPLC fractions (F1mel-F5mel) of 70 tested contained peptides derived from H-2Kb+ CL8-1 melanoma (but not H-2Kb- melanomas) that were capable of conferring susceptibility to CL8-1 TIL lysis on H-2b-expressing target cells (EL4, C1R.Kb), but not on H-2d-expressing P815 target cells. CL8-1 TILs failed to recognize peptides derived from H-2Kb+ nonmelanoma targets such as EL4 or normal B6 splenocytes. Interestingly, CL8-1 TILs appeared to recognize peptide species contained in two HPLC fractions derived from the MCA-105 sarcoma (F1sar and F5sar). Conversely, TILs derived from MCA-105 lesions recognized MCA-105 and CL8-1 tumor cells, as well as F5mel and F5sar peptides presented by EL4 targets. These data support common murine tumor-associated peptide epitopes presented by H-2Kb and recognized by CD8+ CTLs derived from histologically distinct tumors, melanoma and sarcoma.

Animals↗

PubMed1994

Divergent effects of H-2K and H-2D genes on sensitivity of BL6 melanoma cells to NK cells or TNF-mediated cytotoxicity.

Transfection of BL6-8 (H-2Kb-,H-2Db+) and BL6-2 (H-2Kb-,H-2Db-) melanoma clones with H-2Kb or H-2Kd gene resulted in a stable augmentation of their sensitivity to lysis by NK cells or TNF-alpha that was associated with alterations of various phenotypic properties such as loss of endogenous A- and C-type retrovirus production and expression of melanoma-associated antigen and appearance of cell surface carbohydrates reacting with soybean agglutinin (SBA) and Grifonia simplicifolia 1-B4 (GS1B4) lectins. In contrast, transfection of the same clones with H-2Dd or H-2Ld gene did not reverse their resistance to NK cell- and TNF-mediated cytotoxicity and did not affect the phenotype of melanoma cells. These data suggest that the effect of H-2K gene on NK/TNF sensitivity of BL6 cells is indirect and it is closely associated with H-2K-induced phenotypic changes in these cells. To examine the possible role of the observed alterations of cell surface carbohydrates in augmentation of sensitivity of BL6 melanoma cells to lysis by NK cells or TNF, BL6-8 melanoma cells were transfected with cDNA encoding alpha 1,3-galactosyltransferase (alpha 1,3GT). Although the alpha 1,3GT gene-transfected cells expressed alpha-galactosyl epitopes reacting with GS1B4 lectin and reduced sialylation of cell membrane with unmasking SBA lectin-binding carbohydrates, they did not show an increase in tumor cell sensitivity to lysis by NK cells or TNF, indicating that H-2K gene-induced alterations in cell surface carbohydrate expression are not accountable for the observed increase in NK/TNF sensitivity of BL6 melanoma cells. It is possible that the H-2K gene-induced elimination of the endogenous retroviruses might be responsible for the observed increased sensitivity of BL6 melanoma cells to NK cell- and TNF-mediated cytotoxicity.

Animals↗