To examine the role of protein kinase С (PKC) in induction of human colon adenocarcinoma cell line, DETA/W, by polypeptide growth- promoting factors, ornithine decarboxylase activity (ODC) and DNA synthesis were determined in cells depleted of PKC. PKC depletion was achieved by prolonged cultivation (more than 30 passages) with 10⁻⁶ M phorbol 12-myristate 13-acelate. Lack of PKC in studied cells was proved by measurements of PKC activity and immunoreactivity. Although ODC activities and DNA syntheses in PKC-depleted cells were decreased by about 40-50% compared to normal DETA/W cells, the percentage increase of these mitogen-responsive reactions was quantitatively similar in both cell sublines. These results raise the possibility that not all of the biological responses to growth factors are connected with the activation of calcium-dependent PKC.
Human mitochondrial polynucleotide phosphorylase (hPNPase) is an exoribonuclease localized in mitochondria. The exact physiological function of this enzyme is unknown. Recent studies have revealed the existence of a relationship between induction of hPNPase mRNA and both cellular senescence and growth arrest of melanoma cells following β-interferon treatment. The aim of this study was to verify whether the augmented hPNPase mRNA level results in increase of the protein level. In several cell lines established from five metastatic melanoma patients we did not find any such correlation. However, an elevated level of hPNPase protein was observed in interferon-induced HeLa and Jurkat cells. This increase was correlated with a slight shortening of poly(A) tails of mitochondrial ND3 transcript.
Dendritic cells (DC) generated from human umbilical cord blood might replace patients' DC in attempts to elicit tumor-specific immune response in cancer patients. We studied the efficiency of transfection of human cord blood DC with plasmid DNA carrying the enhanced version of green fluorescent protein (EGFP) as a reporter gene, to test if nonviral gene transfer would be a method to load DC with protein antigens for immunotherapy purposes. Cord blood mononuclear cells were cultured in serum-free medium in the presence of granulocyte-monocyte colony stimulating factor (GM-CSF), stem cell factor (SCF) and Flt-3 ligand (FL), to generate DC from their precursors, and thereafter transfected by electroporation. Maturation of DC was induced by stimulation with GM-CSF, SCF, FL and phorbol myristate acetate (PMA). Transfected DC strongly expressed EGFP, but transfection efficiency of DC, defined as HLA-DR+ cells lacking lineage-specific markers, did not exceed 2.5%. Expression of the reporter gene was also demonstrated in the DC generated from transfected, purified CD34+ cord blood cells, by stimulation with GM-CSF, SCF, FL, and tumor necrosis factor α (TNF-α). Transfection of CD34+ cells was very efficient, but proliferation of the transfected cells was much reduced as compared to the untransfected cells. Therefore, the yield of transgene-expressing DC was relatively low. In conclusion, nonviral transfection of cord blood DC proved feasible, but considering the requirements for immunotherapy in cancer patients, transfection of differentiated DC or generation of DC from transfected hematopoietic stem cells provide only a limited number of DC expressing the transgene.
The in vivo effects of some derivatives of aliphatic ketones (2-undecanone, 3-undecanone, 4-undecanone and their derivatives) on L-l sarcoma tumor angiogenesis and VEGF content were studied in Balb/c mice. Mice that inhaled 10% solution of 3-undecanone(3-on) or 1% solution of 2-undecanone propylene acetal (Acpr2) for 3 days after tumor cells implantation, presented lower neovascular response measured by tumor-induced cutaneous angiogenesis test (TIA) and lower tumor VEGF content in 5-days tumors, than non-inhaled controls. Other substances presented various effects on tumor VEGF concentration and angiogenesis. Histological examination of lesions collected from mice inhaled Acpr2, or non-inhaled controls, revealed small diffused areas of necrosis in the former group. In both groups, slight to moderate inflammatory infiltrations were seen at the tumor's margin. In Acpr2 group, there were less small blood vessels at tumor's margin than in the control group.
Background: This is a pilot study evaluating of high-risk melanoma patients (pts) treated with peptide-DC vaccine after lymphadenectomy (LND). DC vaccination was designed to induce the immune response against melanoma antigens in melanoma pts who remain at high risk of dissemination after LND. Methods: DCs were generated from the bone marrow cultured with GM-CSF, SCF, FLT3-L and TNF-a or from peripheral blood adherent monocytes cultured with GM-CSF and IL-4. DCs pulsed with HLA-A2-binding TYR, MART-1 and gp100 peptides and/or HLAA1-binding MAGE-1, MAGE-3 peptides, tumor lysate if available, or with tracer antigen keyhole limpet hemocyanin (KLH), were injected subcutaneously 9 times within 8 months (mos). Boost injections were performed after 12 and 24 mos. Vaccinated pts were matched to unvaccinated controls (22 of 869) by sex, number of metastatic lymph nodes, extracapsular involvement, completion or therapeutic LND, Breslow stage (T), ulceration, and lactate dehydrogenase (LDH) level prior to LND. Results: HLA-A2+, -A1+ or -A3+ melanoma pts (n=22), stage III, N1b-N3, enrolled between Sept. 2002 and Apr. 2004, received 5-16 vaccinations (median: 11) within 2 yrs. Cutaneous delayed type hypersensitivity (DTH) to melanoma peptides was induced in 12 of 22 pts. Peptide-specific IFN-g; producing CD8+ cells were detected in peripheral blood of 13 of 19 pts after vaccination. At least one of these responses to melanoma antigens was elicited in 17 of 22 pts. DTH to KLH was positive in 15 of 22 pts. Eight vaccinated pts are free of disease (follow up is 77-97 mos after LND), and 1 in progression is lost from follow-up by Aug 30, 2010. Survival analysis of vaccinated pts and matched controls is presented in Table 1. Conclusions: The DC/peptide vaccine elicited immune responses to melanoma antigens. Vaccinated pts had clinically substantially longer overall survival (OS) and disease free survival (DFS) than matched control. OS was associated with the immune responsiveness to melanoma antigens and to KLH. Table 1: Survival analysis of vaccinated pts and matched controls. Vaccinated pts (n=22) 3-year OS [%]: 68.2 Matched control (n=22) 3-year OS [%]: 25.7 p-value accounting for matching 0.0290 HR (95% CI)* 3.25 (1.06-9.97) Vaccinated pts (n=19**) 3-year DFS [%]: 40.9 Matched control (n=22) 3-year DFS [%]: 14.5 p-value accounting for matching 0.1083 HR (95% CI)* 2.16 (0.82-5.7) *Cox Proportional Model - Hazard Ratio (HR) of unvaccinated pts **3 pts with recurrence before LND were excluded.