Cytokines IL-1beta, IL-2, IL-6, IL-8, MCP-1, GM-CSF and TNFalpha in patients with epithelial ovarian cancer and their relationship to treatment with paclitaxel.

In vitro work suggests that cytokines may be important modulators of the cytotoxic effects of paclitaxel and subsequent drug resistance. This has been investigated in vivo in patients with ovarian cancer by ELISA. There was consistently elevated expression of IL-6 and IL-8 but not MCP-1, IL-1beta, IL-2, GM-CSF or TNFalpha. Peritoneal fluid concentrations of IL-6, IL-8 and MCP-1 were two to three logs greater than serum concentrations. Elevated concentrations of IL-6 correlated with a poor final outcome (P = 0.039), and increased IL-6 and IL-8 correlated with a poor initial response to chemotherapy (P = 0.041 and P = 0.041, respectively). There was a relatively clear pattern of change in all three cytokines. In serum, IL-6, IL-8 and MCP-1 decreased with the administration of steroids prior to paclitaxel, and increased in the 24 h after paclitaxel. Postoperative drainage fluid was relatively acellular, preventing flow-cytometric analysis of epithelial cells for apoptosis, but suggested activation of T cells by paclitaxel. IL-6 and IL-8 appear to be of prognostic importance in epithelial ovarian cancer. Treatment with paclitaxel is associated with an increase in expression of a limited number of cytokines in patients with ovarian cancer, notably IL-6, IL-8 and MCP-1.

TRAG-3, a novel cancer/testis antigen, is overexpressed in the majority of melanoma cell lines and malignant melanoma.

BACKGROUND: We have identified a novel cancer/testis antigen, TRAG-3, (Taxol Resistance Associated Gene-3) that was initially discovered in search for new genes involved in drug resistance by differential display. Early study of TRAG-3 revealed minimal to absent expression in various normal tissues and over-expression in many carcinoma cell lines including several melanoma lines. MATERIALS AND METHODS: Northern and RT-PCR technologies were used to evaluate TRAG-3 expression in numerous cell lines and tumor tissue. RESULTS: Analysis of a wider panel of normal tissues, 32 melanoma cell lines and 4 malignant melanomas demonstrates TRAG-3 expression in 25 of the 32 melanoma cell lines (78%) and four of four of the malignant melanoma tumors (100%). Of the additional eight normal tissues screened, expression was present in normal testis but absent in all other tissues. RT-PCR evaluation of TRAG-3 reveals two transcripts in many carcinoma cell lines with sequencing of these products demonstrating the 799 bp TRAG-3 transcript and a second alternatively spliced transcript, TRAG-3long TRAG-3 maps to band Xq28 within a MAGE gene complex, however sequence analysis demonstrates that TRAG-3 is not homologous to other known cancer/testis antigens. CONCLUSION: TRAG-3 appears to be a novel cancer/testis antigen.

Discovery of differentially expressed genes associated with paclitaxel resistance using cDNA array technology: analysis of interleukin (IL) 6, IL-8, and monocyte chemotactic protein 1 in the paclitaxel-resistant phenotype.

In an attempt to define the molecular changes associated with the paclitaxel-resistant phenotype in human cancer, a paclitaxel-resistant ovarian cancer cell line, SKOV-3TR, was established through stepwise selection in increasing paclitaxel concentrations. SKOV-3TR was cross- resistant to doxorubicin and vincristine and overexpressed multidrug resistance gene 1 but not multidrug resistance associated protein. SKOV-3TR and the paclitaxel-sensitive SKOV-3 parent line were characterized using human cDNA array technology that examined expression of a wide variety of genes involved in cell growth, signal transduction, cell death, and immune function. cDNA probes from reverse transcribed mRNAs of both paclitaxel-resistant and parent cells were compared to identify genes differentially expressed in the paclitaxel-resistant cells. Of 588 different human cDNA transcripts compared, 6 genes were found to be markedly decreased, and 12 genes increased in the resistant subline. Northern analysis and/or reverse transcription-PCR confirmed that 12 of these 18 genes were over- or underexpressed in SKOV-3TR. In addition, at least eight of the genes were found differentially expressed in several other paclitaxel- and/or doxorubicin-resistant cell lines, both those with increased multidrug resistance expression and those without. Included in the set of overexpressed genes were the cytokines/chemokines interleukin 6, interleukin 8, and monocyte chemotactic protein 1. ELISA assays confirm that mRNA overexpression of these cytokine/chemokines was associated with the increased secretion of these molecules in the tissue culture supernatant. Evaluation of supernatants from an expanded collection of paclitaxel- and Adriamycin-resistant cell lines demonstrated that all of the resistant lines had significant overexpression of at least one cytokine/chemokine as compared with their drug-sensitive parent line. The overexpression of these cytokines seemed to be stable and associated with a drug-resistant phenotype with only a modest induction of cytokine expression in the parent line with short-term paclitaxel exposure. These findings suggest that the development of paclitaxel resistance is accompanied by multiple changes in gene expression including stable alterations in selective chemokine and cytokine expression. The role these associated genetic changes have in the drug-resistant phenotype is discussed.

TRAG-3, a novel gene, isolated from a taxol-resistant ovarian carcinoma cell line.

The mechanisms responsible for the development of the taxol resistance phenotype are unclear, and are likely explained by multiple mechanisms. To understand the molecular changes associated with drug resistance more fully, a taxol-resistant subline, derived from the human ovarian cancer cell line SKOV-3, was established through selection by culture in incrementally increasing taxol concentrations. Comparison of SKOV-3 to SKOV-3TR by differential display identifies a new gene, TRAG-3 (Taxol Resistance Associated Gene- 3). In comparison to the parental line, SKOV-3, TRAG-3 mRNA is overexpressed in the taxol-resistant cell line SKOV-3TR. The nucleotide sequence of the TRAG-3 cDNA contains an open reading frame of 333bp that predicts for a protein product of 110 amino acids. A GenBank search identifies a cosmid clone containing a genomic sequence corresponding to that of TRAG-3. DNA and protein analysis reveals that TRAG-3 has no homology to any known cDNAs or proteins. Northern analysis demonstrates that TRAG-3 is overexpressed in the taxol-resistant breast cancer cell line MDA 435TR as well as the doxorubicin-resistant multiple myeloma cell lines 8226/DOX40 and 8226/MDR10V. A survey of normal tissue shows minimal or absent TRAG-3 mRNA expression. Screening of a wide variety of cancer cell lines demonstrates TRAG-3 expression in many cell lines derived from different tissue types. In summary, TRAG-3 is a novel gene whose expression is associated with the chemotherapy-resistant and neoplastic phenotype.