Bombesin and calcitonin secretion by pulmonary carcinoma is modulated by cholinergic receptors

Three established cell lines derived from human small cell carcinoma of the lung, and known to produce significant amounts of peptide hormones were used to evaluate the regulation of hormone secretion by cholinergic agonists. In two of the cell lines (DMS 53, DMS 153) acetylcholine chloride, bethanechol chloride, and carbamylcholine at the concentrations of 10(-3)M to 10(-5)M stimulated secretion of bombesin and calcitonin as measured by RIA. The third cell line, DMS 406, was not significantly stimulated. Inhibition of induced stimulation by the cholinergic antagonist atropine, but not hexamethonium, indicated the presence of muscarinic rather than the nicotinic type of cholinergic receptors on the stimulatable cells. These receptors appear to mediate hormone secretion comparably to normal endocrine cells.     

Somatostatin 28(1-12) in a somatostatin-secreting human medullary thyroid carcinoma cell line

We have identified a system, the TT human medullary thyroid carcinoma cell line, which we found to contain 31.3 +/- 27.7 ng of somatostatin 28(1-12) immunoreactivity/mg protein. Radioimmunoassay of gel filtration fractions showed that the major form of immunoreactive somatostatin 28(1-12) had a molecular weight of 1,500 daltons. During reversed-phase high pressure liquid chromatography, this 1,500-dalton species coeluted with synthetic somatostatin 28(1-12). Somatostatin 28(1-12) containing forms larger than 7,000 daltons were also observed. Further studies will be required to elucidate the route of processing of prosomatostatin. The fact that the products of prosomatostatin processing in these cells are similar to those in normal tissues indicates that the TT medullary thyroid carcinoma cell line constitutes a useful model for human somatostatin gene expression.     

Somatostatin, but not somatostatin receptor subtypes 2 and 5 selective agonists, inhibits calcitonin secretion and gene expression in the human medullary thyroid carcinoma cell line, TT.

Somatostatin (SRIH) analogs are commonly used to treat symptoms in medullary thyroid carcinoma (MTC), that expresses SRIH receptors (SSTR1 to SSTR5), as does the human MTC cell line TT. The aim of this work was to evaluate whether SRIH, SSTR2 and SSTR5-selective agonists influence calcitonin (CT) secretion and gene expression in the TT cell line. CT secretion was evaluated by chemiluminescence, and gene expression was analyzed by Northern blot. TT cell line proliferation was also assessed by [(3)H] thymidine ([(3)H]thy) incorporation and viable cell number count. SRIH significantly (p < 0.05) reduced [(3)H]thy incorporation (approx. 50 %), viable cell number (approx. 20 %), CT secretion (-30 %) and CT gene expression (approx. 2-fold). Exposure to the SSTR2-selective agonist, BIM-23 120, and to the SSTR5-selective agonist, BIM-23 206, did not modify CT secretion and mRNA levels in TT cells. Thus, SRIH inhibits DNA synthesis, cell proliferation, CT secretion and CT gene expression in the TT cell line, while SSTR2 and 5 selective agonists, although influencing DNA synthesis and cell proliferation, do not modify CT gene expression, suggesting that SRIH may influence gene expression acting through SSTRs other than subtypes 2 and 5. Furthermore, these findings may explain the erratic response of MTC patients in terms of CT plasma levels to treatment with SRIH analogs, like octreotide and lanreotide, which interact mainly with SSTR2 and 5.     

Emergence of p53 mutant cisplatin-resistant ovarian carcinoma cells following drug exposure: spontaneously mutant selection.

We have previously shown that p53 mutations are associated with cisplatin resistance in ovarian carcinoma IGROV-1/Pt 1 cells. The relationship between p53 status and the development of resistance has not been completely elucidated; in particular, the biological mechanisms behind the acquired drug-resistant p53-mutant phenotype were not clearly explained. Thus, in this study, we investigated whether the p53 mutations found in IGROV-1/Pt 1 cells (270 and 282 codons) resulted from selection, under the selective pressure of the cytotoxic treatment, of a spontaneously mutant cell population preexistent in the cisplatin-sensitive parental cell line (IGROV-1) or were induced by drug (genotoxic) treatment. For this purpose, an allele-specific PCR approach was used. Primers carrying the desired mutations (T-->A codon 270, C-->T codon 282) in the 3' terminus, and the corresponding wild-type primers were used to amplify genomic DNA from the original IGROV-1 cell line used to select the mutant IGROV-1/Pt 1. To increase sensitivity, we hybridized blots of the PCRs with the radiolabeled PCR fragment from IGROV-1/Pt 1. Amplification was obtained for IGROV-1 DNA with the mutated allele-specific primers, indicating the preexistence of a mutated population in the IGROV-1 cell line. Titration experiments suggested that the frequency of the mutated alleles was <0.1%. Single-strand conformation polymorphism and allele-specific PCR analysis of the IGROV-1/Pt 0.1 cells, which are less resistant to cisplatin than IGROV-1/Pt 1 cells and which carry both mutant and wild-type p53 alleles with a wild-type predominance, suggested a progressive selection of the mutant population by cisplatin treatment. This is the first observation that indicates that a subpopulation of p53 mutant cells can occasionally be selected by cisplatin treatment. Thus, considering the susceptibility to spontaneous mutations of the p53 gene in advanced ovarian carcinoma, the selection process resulting in emergence of p53 mutant tumors is a possible origin of resistance of ovarian carcinoma to DNA-damaging agents. The survival advantage of p53 mutant cells in the presence of genotoxic agents could be related to a loss of susceptibility to p53-dependent apoptosis and to defects in checkpoints pathways, resulting in genomic instability.     

VEGF expression and its reguration by p53 gene transfection in endometrial carcinoma cells

Vascular endothelial growth factor (VEGF) that activates endothelial cell growth induces angiogenesis, which is indispensable to tumor igenesis and tumor progression. On the other hand, tumor suppressor gene p53 has been considered to regulate VEGF expression, but the detailed relationship between them remains unclear. In this study, we aimed to study VEGF expression in endometrial carcinoma cells and the effect of p53 gene transfection on VEGF expression using p53-mutated endometrial carcinoma cell line, HEC-50B. Immunoblotting for detecting VEGF protein, p53 protein and beta-actin was performed using 11 endometrial carcinoma cell lines. Levels of VEGF in the cultured media were measured by Enzyme immunoassay(EIA). Transfection of wild p53 gene was carried out by SuperFect method in HEC-50B cells, which had mutant p53 gene and did not express p53 protein. The results of immunoblotting were analyzed by NIH image and expressed as values. The results of EIA were expressed as the relative value. The VEGF value was 0.8 +/- 0.3 (n = 6) in p53-wild group, whereas in p53-mutant group it was 1.6 +/- 0.8 (n = 5). VEGF expression was correlated significantly with p53 status (P < 0.05). VEGF levels in p53 gene-transfected cells and the conditioned medium were decreased in 48 hours after p53 gene transfection. VEGF expression was down-regulated by p53 in endometrial carcinoma cells.     

Effect of octreotide on proliferation of in vitro cultured thyroid medullary carcinoma cells

In TT cells, originating from medullary carcinoma of the human thyroid, the presence of receptors for somatostatin was demonstrated at the ultrastructural level. Inhibitory effect of octreotide (a somatostatin analogue) was observed on proliferation of in vitro cultured TT cells and confirmed by evaluating levels of PCNA and Ki-67 proliferation-associated antigens and examining the extent of DNA damage using the comet assay. Our studies indicate a potential for application of somatostatin analogues to diagnosis and adjunct treatment in thyroid medullary carcinomas.     

Abnormal stability of wild-type p53 protein in a human lung carcinoma cell line

We report here that ectopically expressed wild-type p53 protein showed more than 6 times longer half-life than normal human fibroblasts in NCl-H1299, a widely used cell line derived from non-small cell lung carcinoma lacking the expression of p53 protein. We found no abnormality in the phosphorylation and ubiquitination of p53, and the expression levels of MDM2. Although proteasome activity measured in vitro was not significantly different between the tumor cell line and normal human fibroblasts, proteasome inhibitors, ALLN, MG115, and MG132, did not accumulate p53 protein in the tumor cell line, but did accumulate p53 in normal human cells. These results provide a novel mechanism, by which p53 is stabilized in tumor cells, and they suggest that a mediator should exist between ubiquitinated p53 and proteasome, which may be defective in H1299 cells.     

Anti-proliferative effects of a novel isoflavone derivative in medullary thyroid carcinoma: An in vitro study

Currently available treatments for patients with medullary thyroid carcinoma (MTC) with residual or recurrent disease after primary surgery have low efficacy rates. In view of the possible role of estrogen in the development of thyroid neoplasia, we explored whether proliferation of the human MTC TT cell line, might be curbed by carboxy-daidzein-tBoc (cD-tBoc), a novel isoflavone derivative. Estrogen receptor (ER) α mRNA expression in TT cells was more abundant than ERβ, with a ratio of 48:1. Estradiol-17β (E2) increased DNA synthesis in a dose dependent manner. [(3)H]-thymidine incorporation was also stimulated by the ERβ agonist DPN and the ERα agonist PPT. cD-tBoc inhibited TT cell growth as assessed by thymidine incorporation, XTT assay, and microscopic analysis of culture wells. Creatine kinase specific activity, a marker of the modulatory effects of estrogen on cell energy metabolism, was likewise inhibited. The inhibitory effect of cD-tBoc on [(3)H]-thymidine incorporation could be blocked by the ERβ antagonist PTHPP but not by the ERα antagonist MPP, suggesting that the antiproliferative effect of cD-tBoc on these cells is mediated through ERβ. Furthermore, cD-tBoc potently increased apoptosis and cell necrosis. Co-incubation with the antiapoptotic agent Z-VAD-FMK reversed the growth inhibitory effect elicited by cD-tBoc. These results support the hypothesis that estrogens are involved in the proliferation of MTC. The potent anti-proliferative effects mediated by isoflavone derivatives in the human MTC cell line TT suggest and that this property may be utilized to design effective anti-neoplastic agents.     

Effects of wild-type p53 expression on the quantity and activity of topoisomerase IIalpha and beta in various human cancer cell lines.

The p53 null HL-60 cell line was transfected with plasmids coding for either the wild-type p53 or mutant p53 gene. The stable expression of wild-type p53 resulted in a significant increase in sensitivity to the topoisomerase II poisons etoposide and doxorubicin, but not to the topoisomerase II inhibitors razoxane and ADR-529. HL-60 cells expressing wild-type p53 demonstrated 8- to 10-fold more VP-16 induced DNA breaks by the alkaline elution assay. The effect of inducible expression of wild-type p53 was also studied in the p53 null erythroblastoid cell line K562 and in the human squamous carcinoma cell line SqCC. The inducible expression of wild-type p53 in the K562 cell line resulted in a 3-fold increase in sensitivity to VP-16. The quantity of topoisomerase IIalpha was not altered by the transfection as determined by immunoblotting, while the amount of the beta isoform was increased 2.5-fold in HL-60 cells. The topo II catalytic activity present in nuclear extracts was measured as the decatenation of kinetoplast DNA, and found to be unaltered by p53 expression. Immunostaining for topoisomerase IIalpha was substantially diminished in both stable and inducible wild-type p53 expressing cells when three different antibodies were used (two polyclonal and one monoclonal). However, the addition of VP-16 resulted in a rapid appearance of nuclear fluorescence for topoisomerase IIalpha. No changes in topoisomerase IIbeta immunostaining were observed. These results suggest that an epitope for topoisomerase IIalpha is concealed in cells expressing wild-type p53 and that a complex between topoisomerase IIalpha and p53 may be disrupted by the addition of antitumor drugs.     

Comparative study of p53 expression in human carcinoma cell lines A549 and MCF7 under anticancer drug treatment

The effect of anticancer drugs on the expression of p53 protein in tumor cells was studied using the Western Blot analysis. Human lung carcinoma cell line A549 and human breast carcinoma cell line MCF7 sensitive (WT) and resistant (DOX/R) to doxorubicin were used. An increase in p53 protein expression was found in A549 and MCF7 (WT) cells treated with cisplatin, methotrexate, and doxorubicin, whereas the level of p53 was not statistically significantly changed in the MCF7 DOX/R cells. In the untreated MCF7 DOX/R cells the level of p53 protein was markedly higher than in the untreated WT MCF7 cells. A potential role of p53 protein in the development of doxorubicin-resistance in carcinoma cells is discussed.     

Nocodazole-induced p53-dependent c-Jun N-terminal kinase activation reduces apoptosis in human colon carcinoma HCT116 cells

Microtubule-interfering agents are widely used in cancer chemotherapy, and prognostic results vary significantly from tumor to tumor, depending on the p53 status. In preliminary experiments, we compared the expression and phosphorylation profiles of more than 100 protein kinases and protein phosphatases in human colorectal carcinoma cell line HCT116 between p53+/+ and p53-/- cells in response to short term nocodazole treatment through application of Kinetworks immunoblotting screens. Among the proteins tracked, the regulation of the phosphorylation of c-Jun N-terminal kinase (JNK)1/2 at Thr-183/Tyr-185 was the major difference between p53+/+ and p53-/- cells. With the loss of the p53 gene, the levels of phosphorylation of Ser-63 of c-Jun and Thr-183/Tyr-185 of JNK1/2 in p53-/- cells did not increase as markedly as in p53+/+ cells in response to a 1-h treatment with nocodazole or other microtubule-disrupting drugs such as vinblastine and colchicine. Similar observations were also made in MCF-7 and A549 tumor cells, which were rendered p53-deficient by E6 oncoprotein expression. However, arsenate-induced JNK activation in p53-/- cells was preserved. Inhibition of p53 expression by its antisense oligonucleotide also attenuated nocodazole-induced JNK activation in p53+/+ cells. Surprisingly, cotransfection of p53+/+ cells with dominant negative mutants of JNK isoforms and treatment of p53+/+ cells with the JNK inhibitor SP600125 actually further enhanced apoptosis in p53+/+ cells by up to 2-fold in response to nocodazole. These findings indicate that inhibition of p53-mediated JNK1/2 activity in certain tumor cells could serve to enhance the apoptosis-inducing actions of cancer chemotherapeutic agents that disrupt mitotic spindle function.     

Somatostatin receptor subtype 1-selective activation reduces cell growth and calcitonin secretion in a human medullary thyroid carcinoma cell line

Medullary thyroid carcinoma (MTC) is a rare and aggressive tumor and so far medical therapy has provided inconclusive results. In the human MTC cell line TT, expressing all somatostatin (SST) receptor subtypes, cell proliferation decreases with SST and SST receptor subtype 2 (sst(2)), but not sst(5), selective agonist treatment, whereas calcitonin (CT) expression and secretion are reduced by SST, but not by sst(2) and sst(5) agonists. The effectiveness of two new SST analogs, BIM-23926 and BIM-23745, selectively interacting with sst(1), was investigated in the TT cell line. DNA synthesis is significantly reduced by BIM-23926 (27-40% at 10(-10)-10(-6)M) and BIM-23745 (32-90% at 10(-8)-10(-6)M). Viable cell number is also significantly reduced by both BIM-23926 (40% at 10(-12)-10(-6)M) and BIM-23745 ( approximately 40% at 10(-10)-10(-6)M). Treatment with sst(1)-selective agonists significantly reduces CT secretion and gene expression, with a reduction of CREB phosphorylation. These findings suggest that potent sst(1)-selective agonists could have a therapeutic role in MTC.