Pancreatic Cancer Cell Line MIA Research Articles

Retrovirus-mediated herpes simplex virus thymidine kinase gene transfer in pancreatic cancer cell lines: an incomplete antitumor effect.

The transfer of drug-susceptible (suicide) genes to tumor cells by retroviral or adenoviral vectors is a novel approach to the treatment of human tumors. To ascertain the antitumor effect of retroviral transduction of the pancreatic cancer cell lines MIA PaCa 2, CAPAN-1, PANC1, and PSN1 with the herpes simplex virus thymidine kinase (HSV-TK) gene. The vector carried a neoselectable marker gene, the human interleukin-2 gene, an internal ribosome entry coding site, and the region coding HSV-TK. Twenty micromoles or less of ganciclovir did not modify nontransduced TK- cell growth, whereas > or =100 micromol completely inhibited TK- cell growth, indicating that this dosage is cytotoxic per se. The 4 TK- and the 4 transduced cell lines were treated daily with 0.001, 0.01, 0.1, 1, 10, and 20 micromol of ganciclovir for 13 days. CAPAN-1 cell growth was completely inhibited by 0.1 micromol of ganciclovir; higher doses were required to kill PANC1 (10 micromol) and PSN1 (20 micromol). MIA PaCa 2 cell growth decreased following a 20-micromol ganciclovir dosing. The bystander effect was great in the CAPAN-1 cell line and moderate in PANC1; no bystander effect was recorded in MIA PaCa 2 and PSN1 cell lines. Gene therapy with HSV-TK for pancreatic cancer seems effective in only a limited number of tumor-derived cell lines, and this limits its application in vivo.

Molecular cloning and characterization of OSR1 on human chromosome 2p24

During Drosophila hindgut development, bowl, caudal/CDX, brachyenteron/Brachyury/TBX, fork head/FOX, drumstick, lines, and wingless/WNT play important roles. Drosophila bowl gene is homologous to Drosophila odd-skipped (odd) gene and odd-skipped related gene (sob). Here, human OSR1, related to Drosophila odd, was isolated using bioinformatics and cDNA-PCR. OSR1 was found to encode 266 amino-acid protein with three C2H2-type zinc fingers, a tyrosine phosphorylation site (Tyr 203), and several putative PXXP SH3 binding motifs. Three zinc fingers and a tyrosine phosphorylation site were conserved among human OSR1, OSR2, Drosophila odd, sob, and bowl. OSR1 showed 63.6% total amino-acid identity with OSR2. OSR1 gene consisting of three exons was located on human chromosome 2p24. OSR1 mRNA of 2.3-kb in size was detected in adult colon, small intestine, prostate, testis, and fetal lung. OSR1 mRNA was significantly up-regulated in a pancreatic cancer cell line MIA PaCa-2, and was weakly expressed in gastric cancer cell lines OKAJIMA, MKN45, pancreatic cancer cell lines PANC-1, BxPC-3, AsPC-1, PSN-1, Hs766T, and esophageal cancer cell line TE10. Among 10 cases of primary gastric cancer, OSR1 mRNA was up-regulated in 5 cases, and was down-regulated in 2 cases. This is the first report on molecular cloning and characterization of human OSR1.

Aberrant expression and activation of insulin-like growth factor-1 receptor (IGF-1R) are mediated by an induction of IGF-1R promoter activity and stabilization of IGF-1R mRNA and contributes to growth factor independence and increased survival of the pancreatic cancer cell line MIA PaCa-2.

In the present study we investigated the mechanisms responsible for and the biological consequences of the constitutive activation of the insulin-like growth factor-1 receptor (IGF-1R) in the MIA PaCa-2 cells. An aberrant increase in the expression and activation of the IGF-1R was observed during the transition of growth states from exponential to quiescent. The increase in IGF-1R expression is preceded by an increase in IGF-1R mRNA transcript and is associated with an increase in the IGF-1R promoter activity. Inhibition of de novo transcription by actinomycin D increased the stability of IGF-1R mRNA in exponentially growing cells, thereby increasing the expression of IGF-1R to a level similar to that seen in quiescent cells. Increased IGF-1R signaling mediated the growth factor independence of quiescent MIA PaCa-2 cells through the constitutive activation of mitogen-activated protein kinase (MAPK). Exogenous IGF-1 increased cell proliferation and activated MAPK and AKT signaling pathways. The resistance of cells to apoptosis by IGF-1R signaling was mediated through MAPK and phosphatidylinositol 3-kinase (PI3K) pathways and a yet unidentified pathway(s). Thus, aberrant regulation of IGF-1R signaling is required for resistance to apoptosis and growth factor independence of MIA PaCa-2 cells. This likely protects cells from unfavorable conditions and allows cells to rapidly re-enter the cell cycle when conditions are favorable.

CAMP attenuates interleukin-1-stimulated macrophage colony-stimulating factor (M-CSF) expression

Macrophage colony-stimulating factor (M-CSF) is a multifunctional cytokine attributed with key biological functions beyond the first discovered role in promoting proliferation of myeloid cell lineage. The human pancreatic cancer cell line MIA PaCa-2, from which the M-CSF gene was originally cloned, was used to study regulation of M-CSF expression. Expression of M-CSF was inducible by interleukin-1alpha (IL-1alpha), lipopolysaccharide (LPS) and PMA as demonstrated by a biological activity assay, Northern-blot analysis and reverse transcriptase (RT) PCR. Treatment of the cells with forskolin or dibutyryl-cAMP attenuated the expression of M-CSF induced by IL-1alpha or LPS, but not by PMA. Electromobility shift assays showed that IL-1alpha predominantly activated nuclear factor kappaB (NF-kappaB), while PMA preferentially activated activator protein-1 (AP-1). The activation of NF-kappaB, but not AP-1, could be attenuated by cAMP elevation. Relative RT-PCR demonstrated that the expression of a 1.6-kb M-CSF mRNA transcript was more effectively induced by IL-1alpha than a 4.0-kb transcript. By and large the induced expression of both mRNA transcripts could be attenuated by cAMP. M-CSF promoter-driven luciferase reporter-gene assays revealed that cAMP elevation attenuated the IL-1-induced transcription activation of the M-CSF promoter, but it had no effect on PMA-induced transcription. Our findings suggest that cAMP regulates M-CSF gene expression at the transcriptional level and that its inhibitory effect involves NF-kappaB signalling pathway.

Effect of 1-γlinolenyl-3-eicosapentaenoyl propane diol on the growth of human pancreatic carcinoma in vitro and in vivo

Essential fatty acids such as gammalinolenic (GLA) and eicosapentaenoic (EPA) acids have been proposed as anticancer drugs. The aim of this study was to test the effect of a lipid emulsion containing both GLA and EPA in a novel chemical formulation of 1-γlinolenyl-3-eicosapentaenoyl propane diol on the growth of human pancreatic carcinoma in vitro and in nude mice. This compound had a dose-dependent growth-inhibitory effect on human pancreatic cancer cell lines MIA PaCa-2 and Panc-1 in vitro. The concentration necessary for 50% growth inhibition was 25 μmol/l for MIA PaCa-2 and 68 μmol/l for Panc-1 (95% CI 20–29 and 59-77 μmol/l respectively). Nude mice bearing subcutaneous pancreatic tumours produced with the MIA PaCa-2 cell line were treated with the maximum tolerated dose (6.75 mg GLA and 7.3 mg EPA per g of body weight) administered over 10 days by daily intravenous (i.v.) bolus injections. No antitumour effect or major alteration in tumour lipid fatty acid composition was seen in comparison with control animals. Concurrent treatment with parenteral iron (iron saccharate, 5 μg/gram body weight daily) did not make a significant difference. Further improvements in fatty acid delivery mechanisms are necessary before they can become useful anticancer agents.

Inhibitory and stimulatory effects of somatostatin on two human pancreatic cancer cell lines: a primary role for tyrosine phosphatase SHP-1.

Somatostatin (SS-14) and its structural analogue SMS 201-995 (SMS) are recognized as physiological inhibitors of multiple organs and tissue functions through specific membrane receptors (sst1-sst5). The effects of SS-14 and SMS in the growth control of the pancreatic cancer cell lines MIA PaCa-2 and PANC-1 were investigated to identify and clarify the intracellular events involved. In PANC-1 cells, SS-14 and SMS caused inhibition of their basal growth, and that stimulated by epidermal growth factor, with a maximal effect at 0.1-1 microM. To understand the inhibitory mechanisms, we investigated the effects of SS-14 and SMS on phosphotyrosine phosphatase (PTPase) activity and, more specifically, that of tyrosine phosphatase SHP-1 (PTP1C). SS-14 and SMS caused significant increases in total cellular PTPase activity, and particularly SHP-1, with maximal activation within 1 min. Inhibition of membrane tyrosine kinase and p42 MAP kinase activities was also observed, in response to SS-14 and SMS. In MIA PaCa-2 cells, SS-14 and SMS were associated with a positive growth response at 1-10 nM, after 4 days of culture in serum-free medium. Total cellular PTPase activity was slightly increased, but SHP-1 activity could not be detected; its absence in this cell line was confirmed by Western blot. Membrane tyrosine kinase activities were significantly increased by SS-14 and SMS at concentrations needed for maximal growth. p44/p42, which are constitutively active in this cell line, and p38 activities were not affected by somatostatin. In conclusion, somatostatin can exert different effects on human pancreatic cancer cell growth, depending upon the presence or absence of SHP-1. This enzyme can play a key role in the control of cell proliferation, and its cellular presence may determine the therapeutic potential of somatostatin in the control of cancer cell growth.

Enhanced Cytotoxicity of Nucleoside Analogs by Overexpression of Mitochondrial Deoxyguanosine Kinase in Cancer Cell Lines

The cytotoxic anti-cancer purine nucleoside analogs 2-chloro-2'-deoxyadenosine (CdA), 9-beta-D-arabinofuranosylguanine (araG), and 2',2'-difluorodeoxyguanosine (dFdG) are phosphorylated by human mitochondrial deoxyguanosine kinase (dGK) in vitro. We overexpressed dGK as a fusion protein to the green fluorescent protein in the human pancreatic cancer cell lines PanC-1 and MIA PaCa-2 to determine the importance of dGK-mediated nucleoside analog phosphorylation. The transfected cells showed mitochondrial fluorescence patterns, and the mitochondrial locations of endogenous and overexpressed dGK were verified by Western blot analysis of cell extracts with polyclonal anti-dGK antibodies. The increase of dGK activity in the overexpressing cells was approximately 4-fold. These cell lines exhibited increased sensitivity to CdA, araG, and dFdG as compared with the untransfected parent cell lines. This is, to our knowledge, the first demonstration of a correlation between the activity of a mitochondrial deoxyribonucleoside kinase and the cytotoxicity of nucleoside analogs. Our data imply that the dGK activity is rate-limiting for the efficacy of nucleoside analogs in the cell lines investigated.

Interleukin-8 can mediate acute-phase protein production by isolated human hepatocytes.

During the course of studies designed to identify the role of cytokines in the reprioritization of hepatic protein synthesis associated with cachexia we detected a hepatocyte-stimulating moiety in the supernatants of pancreatic cancer cells that was unrelated to interleukin (IL)-6. This study identifies that moiety as IL-8 and investigates the role of IL-8 in the induction of acute-phase protein production. The human pancreatic cancer cell line MIA PaCa-2 produced >1 ng/ml of IL-8 per 24 h, and supernatants from this cell line induced C-reactive protein (CRP) production from isolated human hepatocytes. Addition of neutralizing anti-human IL-8 antibody to such supernatants produced almost complete inhibition of CRP production. The addition of recombinant human IL-8 to hepatocytes resulted in a dose-dependent increase in CRP, alpha1-acid glycoprotein, and alpha1-antichymotrypsin production and a decrease in the production of transferrin and prealbumin. This study demonstrates that recombinant or tumor-derived IL-8 can modulate acute-phase protein production from isolated human hepatocytes and from human hepatoma cells.

Efficacy of p120 antisense-mediated therapy for pancreatic cancer

p120 Antisense oligodeoxynucleotides were used to determine whether they inhibited cell growth of MIA PaCa-2, a highly tumorigenic human pancreatic carcinoma cell line. Growth inhibition assays were determined in vitro by the ability of these oligomers to inhibit DNA synthesis and cell growth. For in vivo studies, nude mice were injected with cells and palpable tumors were found in 16 of 20 animals by day 14. Sixteen animals (8 in each group were then treated daily (25 mg/kg intraperitoneally) for up to 40 days with nonsense control oligomers or p120 antisense oligomers. p120 Antisense oligomers inhibited the in vitro proliferation of MIA PaCa-2 cells in a dose-dependent manner, and optimal growth inhibition of greater than 90% was achieved at an antisense oligomer concentration of 100 μmol/L. The tumor volume was calculated for antisense- and nonsense-treated animals. Fifteen days after the beginning of treatment, control animals had a significantly greater ( P = 0.0035) tumor volume (425 ± 244 mm 3 above baseline) as compared to p120 antisense-treated animals (166 ± 116 mm 3). Seven of the eight control animals formed tumors that had a volume greater than 1200 mm 3 45 days after treatment was begun, whereas only three of eight p120 antisense-treated animals had tumors that were this large. Two of the latter three animals had relatively large, palpable tumors (150 mm 3) prior to treatment. Twenty days after treatment was stopped (day 60), all animals had tumors larger than 1200 mm 3. p120 Antisense oligomers were effective for inhibiting in vitro growth of the pancreatic cancer cell line MIA PaCa-2. In preliminary studies, p120 antisense oligomers appeared to inhibit the rate of growth in nude mice; however, no cures were achieved. The most effective response was seen in animals with initial low tumor burden.

Cell cycle arrest and induction of apoptosis in pancreatic cancer cells exposed to eicosapentaenoic acid in vitro.

Eicosapentaenoic acid (EPA) has been shown to have an inhibitory effect on the growth of several pancreatic cancer cell lines in vitro. This study investigates the mechanism of growth inhibition and cytotoxicity of EPA on the pancreatic cancer cell line MIA PaCa-2. Cells were analysed for cell count, viability, cell cycle distribution and ultrastructural changes. There was a time- and dose-dependent decrease in cell count and viability in cultures of pancreatic cancer cells supplemented with EPA. Flow cytometric DNA analysis of MIA PaCa-2 cells incubated with EPA demonstrated the presence of sub G1 populations corresponding to the presence of apoptotic cells and the blockade of cell cycle progression in S-phase and G2/M-phase. The presence of apoptosis in EPA-supplemented cultures was further confirmed by DNA fragmentation and ultrastructural changes associated with apoptosis. Therefore, we conclude that EPA mediates its effect on the pancreatic cancer cell line MIA PaCa-2, at least in part, via cell cycle arrest and the induction of apoptosis.

Two subclasses of EGF receptors in the human pancreatic cancer cell lines CAPAN-1 and MIA PaCa-2

The existence of different classes of EGF receptors in human pancreatic cancer cells has yet not been determined. EGF binding to two cancer cell lines (CAPAN-1 and MIA PaCa-2) was studied. Two classes of EGF binding sites were characterized. The first class of EGF binding sites demonstrated a high affinity and low capacity for EGF, with a Kd of 0.25 +/- 0.11 nM, close to the concentration of EGF suggested to be present in human pancreatic juice. The second class of EGF binding sites had a lower affinity and a higher capacity for EGF, with Kd of 1.78 +/- 0.61 nM. The total number of EGF binding sites was about 40,000/cell. Treatment of the cells with a phorbol ester, TPA, caused a complete loss of the high affinity binding sites and also caused a decrease in the concentration of the lower affinity binding sites present on the cells. Interestingly, with the increasing age of the cells, the concentration of both the high and low affinity EGF binding sites was significantly decreased. In the presence or absence of fetal calf serum, EGF, at concentrations higher than 1.10(-10)M, exerted a dose-dependent mitogenic effect on the growth of the pancreatic cancer cells in culture. These data demonstrate the existence of two classes of binding sites for EGF on some human pancreatic cancer cells and a possible role of EGF in the growth of pancreatic tumors.