Adhesion polypeptides are useful for the prevention of peritoneal dissemination of gastric cancer

We examined the effect of adhesion polypeptides on the adhesion and invasiveness of gastric cancer cell lines. We previously reported the establishment of an extensively peritoneal-seeding cell line, OCUM-2MD3, from a poorly seeding human scirrhous gastric carcinoma cell line, OCUM-2M. Both 21 and 31 integrin expression was markedly increased on OCUM-2MD3 cells compared with OCUM-2M cells, and the ability of OCUM-2MD3 cells to bind to the extracellular matrix (ECM) was also significantly higher than that of OCUM-2M cells. The adhesion polypeptides, YIGSR and RGD, and two RGD derivatives significantly inhibited the adhesion of OCUM-2MD3 cells to the submesothelial ECM, while not inhibiting the adhesiveness of OCUM-2M cells and two well differentiated human gastric cell lines, MKN-28 and MKN-74. The YIGSR and RGD peptides also significantly inhibited the invasiveness of OCUM-2MD3 cells. The survival of nude mice with peritoneal dissemination given YIGSR sequenc e intraperitoneally was obviously longer than that of untreated mice. The survival of mice treated with RGD was also improved, and this effect was increased using the RGD derivatives, poly(CEMA-RGDS) and CM-chitin RGDS. These polypeptides appear to block the binding of integrins, which are expressed on OCUM-2MD3 cells, to the submesothelial ECM, and consequently inhibit peritoneal implantation. The peritoneal injection of adhe-sion polypeptides may be a new therapy against the dissemination of scirrhous gastric cancer, and may be useful for the prevention of dissemination in high-risk patients. © Rapid Science Ltd.     

Establishment of lymph node metastatic model for human gastric cancer in nude mice and analysis of factors associated with metastasis

The actual mechanisms responsible for lymph node metastasis in gastric cancer are still unclear. To investigate the mechanisms of lymph node metastasis in gastric cancer, we established a lymph node metastatic model for human scirrhous gastric carcinoma. Lymph node metastasis had frequently developed after orthotopic implantation of OCUM-2M LN derived from a scirrhous gastric cancer cell line, OCUM-2M, which had low capacity for lymph node metastasis. We elucidated the different characteristics including binding ability, migratory capacity and immunoresponses induced by the cell surface molecules of these two cell lines. The binding ability to Matrigel and migratory capacity of OCUM-2M LN cells were significantly greater than those of OCUM-2M cells. On flow cytometric analysis, both OCUM-2M and OCUM-2M LN cells strongly expressed HLA-I (99.5 and 97.1%) and LFA-3 (76.6 and 99.2%) in level of expression between the two cell lines, but neither cell line expressed HLA-II (0.0 and 0 .0%), B7-1 (0.0 and 0.0%) or B7-2 (0.4 and 0.3%). ICAM-1 expression in OCUM-2M LN cells was weaker (0.7%) than that in OCUM-2M cells (36.8%). Strong adhesiveness and cytotoxicity of mononuclear lymphocytes for OCUM-2M cells were observed in adhesion and cytotoxic assays, both of which were significantly decreased by the addition of anti-ICAM-1 antibodies. On the other hand, the adhesiveness and cytotoxicity of OCUM-2M LN cells were significantly less than those of OCUM-2M cells, and were not affected by the addition of anti-ICAM-1 antibodies. These findings suggest that decreased ICAM-1 expression in a new gastric cancer cell line with a high rate of lymph node metasta-sis may in turn decrease immune responses mediated through LFA-1-dependent effector cell adhesion, and that this escape from the immunosurveillance system may be one of the factors inducing lymph node metas-tasis. In conclusion, we established a gastric cancer cell line, OCUM-2M LN, with a high rate of lymph node metasta sis. An in vivo lymph node-metastatic model with this cell line should be useful for analysing the mech-anism and therapeutic approach of lymph node metastasis. © Rapid Science Ltd.     

Lipid-mediated gene transfection of intercellular adhesion molecule-1 suppresses the peritoneal metastasis of gastric carcinoma

We have previously reported that a decreased expression level of ICAM-1 in cancer cells frequently led to the development of lymph node metastasis, and suggested that ICAM-1 gene transfection may inhibit lymph node metastasis. In the present study, we investigated whether ICAM-1 gene therapy for the peritoneal metastasis of gastric carcinoma is useful as a new immuno-gene therapy. ICAM-1 gene was transfected into a gastric cancer cell line, OCUM-2MD3 (2MD3), which has a high metastatic ability to the peritoneum. A transfectant cancer cell line, 2MD3/ICAM-1, had high ICAM-1 expression on the cell surface. The adhesion and cytotoxicity abilities of peripheral blood mononuclear cells were significantly increased against 2MD3/ICAM-1 cells in comparison with 2MD3 cells. Mice inoculated with 2MD3/ICAM-1 cells in the peritoneal cavity had a significantly better survival rate than those inoculated with 2MD3 cells (log-rank test, p<0.05). Histologic findings revealed that more mononuclear cells existed around the metastatic nodules in 2MD3/ICAM-1. Although gastric carcinoma frequently causes peritoneal metastasis, no useful therapy for the metastasis of gastric carcinoma has been developed. These findings revealed that ICAM-1 gene transfection to cancer cells could be a useful immuno-gene therapy for the peritoneal metastasis of gastric carcinoma.     

Macrophage Priming and Activation During Fibrosarcoma Growth: Expression of c-myb,c-myc,c-fos,and c-fms

Macrophages (M?)³ function by a two-step process that includes priming (induction of cytokine and enzyme mRNA) and activation (production of effector molecules). The initial steps in M? priming involve the expression of certain proto-oncogenes that regulate expression of other genes. Because tumor growth primes M? to produce several suppressor monokines, we determined if cancer induced M? expression of these proto-oncogenes. Unstimulated peritoneal M? from tumor-bearing hosts (TBH) constitutively expressed the proto-oncogenes c-fms, c-fos, c-myc, and c-myb, whereas normal host (NH) M? had little or no expression of these proto-oncogenes. When M? were given a 24-h adherence priming stimulus, NH M? expressed c-fms and c-fos at levels equivalent to TBH M? constitutive expression. Adherence had little or no effect on c-fms and c-fos expression in TBH M? or on NH and TBH M? c-myc expression. c-myb expression was not induced in NH M? during adherence and was strongly decreased in TBH M?. Activation with a 1-h lipopolysaccharide-treatment increased NH and TBH M? expression of c-fms, c-fos, and c-myc, with higher expression of these proto-oncogenes in TBH M?. Activation failed to induce c-myb expression in NH M? and completely inhibited expression in TBH M?. Because c-fms, c-fos, and c-myc are normally expressed early during M? activation, our results suggest that tumor growth primes M? by inducing expression of these proto-oncogenes. c-myb is expressed in immature M? and is downregulated during M? activation. These observations explain why NH M? expression of c-myb was not induced and are consistent with reports that suggest TBH M? have not reached full developmental maturity. The induction of M? protooncogene expression during cancer may put M? in a primed state, which leads to earlier and stronger production of adverse suppressor and cytotoxic molecules.     

Expression of platelet-derived growth factor and c-myc in atherosclerotic lesions in cholesterol-fed chickens: immunohistochemical and in situ hybridization study

Immunohistochemical examination showed no significant expression of platelet-derived growth factor-A (PDGF-A), PDGF-B, PDGF receptors, or of c-myc in the thoracic and abdominal aortas of normal roosters. In cholesterol-fed roosters, intense immunohistochemical reaction for PDGF-B, PDGF receptor, and c-myc was seen in the lipid-rich thickened intimal lesions of the thoracic and abdominal aortas while no significant immunoreaction for PDGF-A was demonstrated in the same lesions. In accordance with immunohistochemical findings, in situ hybridization demonstrated a significant level of expression of PDGF-B, PDGF-A receptor, PDGF-B receptor, and c-myc genes in proliferating intimal cells of the thoracic and abdominal aortas. These results suggest that coordinate actions of PDGF-B and c-myc play an important role in proliferation of intimal cells in the developing atherosclerotic lesions in chickens.     

No correlation of c-myc overexpression and p53 mutations in liposarcomas

 Although it is well known that oncogenesis is a multistep process involving the activation of normal cellular genes to become oncogenes and/or the inactivation of tumor suppressor genes, this process has seldom been investigated in soft tissue tumours. We screened a group of 36 liposarcomas for genetic abnormalitis in the p53 tumour suppressor gene and c-myc oncogene. Altered c-myc gene expression was examined by differential RT-PCR assay. p53 Gene mutations in exons 4–8 were analysed by using PCR-SSCP analysis and direct sequencing. Elevated c-myc expression was found in 6 of 31 liposarcomas (19.4%). p53 Gene mutations were observed in 5 of 36 liposarcomas (13.9%). Both genetic alterations were associated with the histological subtype of liposarcomas. Whereas c-myc gene expression was a characteristic of myxoid/round cell liposarcomas, p53 gene mutations were found more frequently in pleomorphic variants. Liposarcomas of the well-differentiated subtype showed neither p53 gene mutations nor altered c-myc gene expression. Our results indicate that the c-myc oncogene and the p53 tumor suppressor gene do not seem to cooperate in the oncogenesis of liposarcomas. Received: 22 April 1998 / Accepted: 11 May 1998     

Increased expression of alpha2beta1-integrin in the peritoneal dissemination of human gastric carcinoma

The prognosis of advanced gastric cancer remains poor, given the frequent incidence of peritoneal metastasis. beta1-integrin is known to be associated with metastasis, though few reports have addressed the expression of beta1-integrin subunits in gastric cancer at primary and peritoneal lesions from the perspective of individual cases. We studied specimens from primary tumors from 50 patients and from metastatic peritoneal lesions from 27 patients with gastric carcinoma, including specimens from 22 metastatic lesions taken from the same patients whose primary tumors were sampled. Expression of beta1-integrin subunits, alpha2-alpha6beta1 integrins, was studied using an immunohistochemical method. alpha2beta1-integrin was significantly expressed on a larger proportion of tumor cells in peritoneal metastasis (70.4%) than in primary tumors (48%) (p<0.05), though alpha3beta1, alpha4beta1, alpha5beta1 and alpha6beta1-integrins did not demonstrate significant discrepancy. The expression of alpha2beta1-integrin in peritoneal lesions was significantly increased compared with its expression in the primary lesion in the same individual. In contrast, no relationship was found between the expression level of beta1 integrins and clinicopathological parameters. Peritoneal implantation of gastric carcinoma might be closely associated with alpha2beta1-integrin.     

Analysis of c-myc DNA amplification in non-small cell lung carcinoma in comparison with small cell lung carcinoma using polymerase chain reaction

Previous studies of c-myc DNA amplification in lung cancer have focused primarily on analysis of small cell carcinoma or its tumor cell lines. There are few data about c-myc DNA amplification in histological types of lung cancer other than small cell carcinoma. Therefore the present study was conducted to investigate c-myc oncogene amplification in non-small cell lung carcinoma. We studied 46 lung tumor specimens for c-myc DNA amplification (15 adenocarcinomas, 15 squamous cell carcinomas, 6 large cell carcinomas, and 10 small cell carcinomas). Polymerase chain reaction, digoxigenin DNA labeling, and elecrophoresis were utilized to investigate the c-myc copy number in the lung tumor specimens. The c-myc copy number of non-small cell carcinoma ranged from 1.5 to more than 20.0 in adenocarcinoma and squamous cell carcinoma, and from 6.0 to 12.0 in large cell carcinoma. That of small cell carcinoma ranged from 1.8 to 12.0. The c-myc copy number of non-small cell carcinoma was significantly higher that than of small cell carcinoma (Wilcoxon rank sum test, Z=2.06, P=0.040). However, the differences in c-myc copy number among these four histological types were not statistically significant. Amplification of c-myc (more than 4 copies) was observed not only in small cell carcinoma but also in non-small cell carcinoma at similarly high frequency (12/15 in adenocarcinoma and squamous cell carcinoma, 6/6 in large cell carcinoma, and 9/10 in small cell carcinoma). Received: 17 October 2000 / Accepted: 7 May 2001     

Expression of c-myc protein is related to cell proliferation and expression of growth factor receptors in transitional cell bladder cancer

Archival biopsy specimens from transitional cell bladder tumours (n=185) were analysed immunohistochemically for expression of c-myc protein. The results were compared with compared with histopathological and clinical parameters and survival. Forty-three per cent of the tumours were negative for c-myc protein and weak, moderate, or strong cytoplasmic expression was found in 34, 14, and 9 per cent of cases, respectively. Nuclear positivity for c-myc protein was detected in 35 per cent of tumours and nuclear opositivity was related to overexpression of c-erb B-2 (P=0.01) and a high proportion of nuclei were also positive for p53 oncoprotein (p<0.05). Cytoplasmic expression of c-myc protein was related to histological grade (P=0.005), papillary status (P=0.007), the S-phase fraction (P=0.008), the mitotic index (P=0.021), overexpression of epidermal growth factor receptor (P=0.045), and c-erb B-2 (P=0.17). Expression of c-myc protein was not significantly related to the progression of tumours and it had no prognostic value in survival analysis. Independent predictors were the T-category (P<0.001), papillary status. (P=0.001), and S-phase fraction (P=0.061). The results show that while c-myc gene product participates in growth regulation of human bladder cancer cells, it has no independent prognostic significance.     

Expression of the c-myc and Ca-ATPase genes in cardiac muscle during adaptation to repeated stress

In the course of adaptation to repeated stress, the expression of the proto-oncogene c-myc found to increase much more rapidly than that of the Ca-ATPase gene. It is suggested that an increase in the level of c-myc expression may activate the structural Ca-ATPase gene and possibly also the heat-shock proteins. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 117, N ^o 2, pp. 124–126, February, 1994     

Analysis of c-fos,c-jun,c-erbB1, c-erbB2 and c-myc in primary lung carcinomas and their lymph node metastases

The purpose of this study was to identify possible alterations in proto-oncogenes (c-fos, c-jun, c-erbB1, c-erbB2 and c-myc) at the protein level in primary lung carcinomas and simultaneous metastatic lymph nodes of 21 patients. The analysis showed that proteins of c-jun and c-myc were expressed in a significantly higher frequency in metastases than in primary lung tumors. Gross differences were not found between primary tumors and metastatic tumors with regard to the expression of c-erbB1, c-erbB2 and c-fos. The finding of cases with a higher expression of c-jun and c-myc in lymph nodes suggests that metastatic capability may be higher in certain cell populations.     

c-myc GENE ABNORMALITIES IN MUCOSA-ASSOCIATED LYMPHOID TISSUE (MALT) LYMPHOMAS

c-myc gene abnormalities associated with lymphomagenesis, including rearrangements and mutations in the regulatory region between exon I and intron I, have been studied in 54 MALT lymphomas (43 low and 11 high grade) and 36 nodal lymphomas (27 low and 9 high grade). By Southern blot analysis, none of the 54 MALT lymphomas but 2 of 36 nodal lymphomas had c-myc gene rearrangements. Defined tumour cell populations from all MALT lymphoma cases were isolated by microdissection from frozen tissue sections and analysed by polymerase chain reaction–single-strand conformational polymorphism (PCR–SSCP) and direct sequencing for somatic mutations in the exon I/intron I region of the gene. Point mutations in this region were identified in nine cases of MALT lymphoma (7/43=16·2 per cent of low grade; 2/11=18·1 per cent of high grade). These mutations were located at either the exon I/intron I border of myc intron factor (MIF) binding sites, which are critical in the negative regulation of c-myc expression. Of the nodal lymphomas, only the two cases (5·6 per cent) with c-myc gene rearrangement showed scattered or clustered mutations. These results suggest that c-myc mutations in MALT lymphomas are unlikely to be associated with chromosome translocation, which is the main cause of somatic mutations observed in other types of lymphoma. The mutations involving the c-myc regulatory regions may play a pathogenetic role in at least a proportion of MALT lymphomas. © 1997 by John Wiley & Sons, Ltd.     

Role of c-myc and CD45 in spontaneous and anti-receptor-induced apoptosis in adult murine B cells

Although adult murine B cells can be stimulated to proliferateby IgM receptor cross-linking, we and others have shown thatthese cells will undergo apoptosis in vitro in a dose-, time-and temperature-dependent manner with polyclonal but not monoclonalantl-lgM. To test the role of c-myc and cell cycle progressionin B cell apoptosis, we examined normal, Sp6 antl-TNP Ig andEµ-myc transgenic splenocytes for receptor-mediated apoptosisin vitro. In normal mice, both spontaneous and anti-lgM-lnducedprogrammed cell death were specifically blocked by antisenseoligodeoxynucleotides for the c-myc proto-oncogene, whereasnonsense myc oligonucleotides and irrelevant oligonucleotideshad only a minor effect. Similarly, TNP-dextran-induced apoptosisin Sp6 antl-TNP transgenics was inhibited by antisense c-myc.This effect was not due to the mitogenic effects of unmethylatedCpG-contalnlng sequences because ones lacking this motif, aswell as methylated oligonucleotides containing this motif, preventedapoptosis, and mitogenic doses of lipopolysaccharlde failedto Inhibit anti-lgM-driven cell death. Importantly, antisensec-myc also prevented the anti-lgM-lnduced increase in myc proteinspecies. Moreover, spontaneous apoptosis in vitro was exaggeratedin Eµ-myc transgenic B cells. To examine the role of CD45in anti-lgM-induced apoptosis, we treated spleen cells fromCD45 knockout mice, which do not proliferate with anti-IgM,and found that B cells from these underwent apoptosis normallydespite the lack of entry into S. These data suggest that anti-IgMdriven apoptosis does not require CD45. Rather, apoptosis maybe due to an overexpression of myc protein in the absence ofsignals which can drive B cells productively Into S, but thefailure to proliferate normally is insufficient for apoptosisto occur.     

Expression of oncoproteins in primary human non-small cell lung cancer and incidence of metastases

In the current study the relationship between the incidence of metastatic spread and expression (at the protein level) of various proto-oncogenes was investigated in 217 human non-small cell lung carcinomas. Tumors with an overexpression of proteins encoded by the oncogenes c-jun and c-myc showed a significantly increased formation of metastases (c-jun: P = 0.008; c-myc: P = 0.018). No significant correlations were found between the expression of the c-fos, c-erbB1, c-neu and c-ras products and metastatic spread.     

Suppression of serum-induced c-jun expression by activated Ki-ras in human colon cancer cells

Summary Through gene-targeting, we have established human colon cancer cell lines, HK2-6 and HKe-3, with and without activated Ki-ras, respectively, derived from a human colon cancer cell line HCT116, and we have reported that activated Ki-ras is involved in the deregulation of c-myc expression. To further examine the relation between Ki-ras-mediated signals and other immediate early genes, c-jun was analyzed on these cells stimulated by serum. Rapid and strong induction of c-jun was observed in HKe-3, but not in HCT116 or HK2-6. To elucidate the regulatory mechanisms of c-jun expression by Ki-ras, protein kinase C (PKC) and c-Raf were examined at serum-starved and serum-stimulated conditions. Phosphorylations of c-Raf were same among these cells, however, the cytosolic PKC activity in HKe-3 was two times higher than that in HCT116 on serum-starved and serum-stimulated conditions. These results suggested that serum responsiveness of c-jun may be suppressed by activated Ki-ras through PKC rather than c-Raf pathway in colon cancer cells.     

c-myc null cells misregulate cad and gadd45 but not other proposed c-Myc targets

We report here that the expression of virtually all proposed c-Myc target genes is unchanged in cells containing a homozygous null deletion of c-myc. Two noteworthy exceptions are the gene cad, which has reduced log phase expression and serum induction in c-myc null cells, and the growth arrest gene gadd45, which is derepressed by c-myc knockout. Thus, cad and gadd45 are the only proposed targets of c-Myc that may contribute to the dramatic slow growth phenotype of c-myc null cells. Our results demonstrate that a loss-of-function approach is critical for the evaluation of potential c-Myc target genes.