Preferential localization of c-kit product in tissue mast cells,basal cells of skin,epithelial cells of breast,small cell lung carcinoma and seminoma/dysgerminoma in human: immunohistochemical study on formalin-fixed,paraffin-embedded tissues

Eighteen hundred and eighty-four cases of human solid tumours and 833 samples of normal human tissues, formalin-fixed and paraffin-embedded, were examined immunohistochemically for expression of c-kit oncogene product using polyclonal antibody against synthesized c-kit peptide. Seminoma/dysgerminoma and small cell lung carcinoma (SCLC) show preferential c-kit expression at 92% and 36% frequency, respectively, whereas only sporadic cases of cervical carcinoma and non-SCLC lung carcinoma show c-kit positivity. A normal tissue counterpart positive for c-kit product is detected in the testis (spermatocyte) and ovary (oocyte) but not in the lung or the cervix. In contrast, normal epithelial cells of the breast, skin basal cells and tissue mast cells harbour c-kit product, but transformed cells of the former two are largely deficient in the c-kit protein. One hundred and thirty-nine neuroendocrine tumours and 39 non-pulmonary small cell carcinomas were all negative, except for two cases of neuroblastoma. This indicates a distinct character for SCLC in c-kit expression. The c-kit product may be a useful marker in diagnostic pathology of seminoma/dysgermona and SCLC among human solid tumours, and in distinction of SCLC from non-pulmonary small cell carcinoma.     

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.     

Effect of hepatocyte growth factor on the expression of E- and P-cadherin in gastric carcinoma cell lines

Hepatocyte growth factor (HGF), identical to scatter factor, (SF) is a secretory glycoprotein from fibroblasts which dissociates and increases the motility of various types of epithelial cells. After treatment of three gastric carcinoma cell lines (MKN-28, MKN-45 and TMK-1) with HGF (10 ng/ml), TMK-1 cells lost their tight cell to cell contact and showed marked scattering, while the two other cell lines remained unaffected. To learn about the underlying mechanism of the HGF induced scattering, we examined the expression of adhesion molecules and growth factor/receptor systems at the mRNA and protein level. The observed scattering of treated TMK-1 cells was associated with a reduction in the expression of E- and P-cadherin protein. The respective mRNA levels remained unchanged after HGF/SF treatment. In the two other cell lines, which showed no scattering, there were no changes in the expression of E-and P-cadherin. All other growth factors and their receptors examined (TGF-, EGFR, c-met and c-erbB₂) remained constant and were not affected by HGF treatment. The results suggest that HGF/SF may regulate cell adhesion in gastric carcinomas via E-and P-cadherin expression at the protein level.     

Different tumorigenicity of cell clones derived from stromal fibroblasts of human colon cancer xenografts

The tumorigenicity of cell clones derived from fibroblast lines isolated from colon cancer xenografts is studied in thymus-free animals. During cloning of the cell line obtained from the 3rd passage of the xenograft about 20% of the clones proved to be nontumorigenic, whereas such cells were not found in the line obtained from the 89th passage. Cytogenetic analysis of nontumorigenic clones revealed monosomy for the 13th chromosome with no alterations in the other chromosome pairs. Hybridization for the presence of Alu sequences was negative. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, N ^o 8, pp. 184–187, August, 1994 Presented by Yu. N. Solov'ev, Member of the Russian Academy of Medical Sciences     

Differential modulation of proliferation, matrix metalloproteinase expression and invasion of human head and neck squamous carcinoma cells by c-erbB ligands

Evidence suggests that there is an association between the abnormal expression of members of the c-erbB receptor tyrosine kinase family and poor prognosis in head and neck squamous cell carcinomas (HNSCC). Until now, the relative contributions of different c-erbB ligands to HNSCC progression have not been clearly defined. In this paper we examined the effects of ligands with different c-erbB receptor specificities in terms of their stimulation of HNSCC proliferation, expression of matrix metalloproteinases (MMPs) and invasion. Heregulin-beta1 (HRG-β1; selective c-erbB3/B4 ligand) was found to stimulate proliferation in the majority of cell lines, whereas epidermal growth factor (EGF; EGFR ligand) and betacellulin (BTC; EGFR/B4 ligand) induced variable responses. All three ligands up-regulated multiple MMPs including collagenases, stromelysins, matrilysin and gelatinase B (MMP-9) but had minimal or no effects on gelatinase A (MMP-2), MT1-MMP and tissue inhibitors of MMPs (TIMPs). MMP-9 mRNA was induced to a higher level than other MMPs, although with slower kinetics. HRG-β1 was less active than EGF and BTC at the optimal concentration (relative potency of EGF:BTC:HRG = 3:4:1). In vitro invasion through Matrigel was also increased by all three ligands in proportion to their MMP up-regulation. A specific anti-EGFR monoclonal antibody (mAb ICR62) inhibited MMP up-regulation, migration and invasion induced by all three ligands, whereas an anti-c-erbB-2 mAb ICR12 inhibited mitogenic and motogenic responses following ligand stimulation but had no effect on MMP expression. These results suggest that c-erbB ligands may differentially potentiate the invasive phenotype of HNSCC via co-operative induction of cell proliferation, migration and proteolysis. The EGFR signalling pathway appears to be the dominant component controlling the proteolytic and invasive phenotype in HNSCC, whereas the c-erbB-2 signalling pathway is responsible, in part, for the mitogenic and motogenic effects of ligands. This revised version was published online in July 2006 with corrections to the Cover Date.     

Characteristics of rhabdomyosarcoma cell lines derived from uterine carcinosarcomas

 Rhabdomyosarcoma (RMS) is occasionally found in the female genital tract, and mostly appears as one of the heterologous mesenchymal components in uterine carcinosarcoma designated as malignant mixed müllerian tumour (MMMT). We examined the biological properties of a pure rhabdomyosarcoma (RMS) cell line designated FU-MMT-3, which was newly established from a surgical specimen taken from a patient with uterine MMMT. We also evaluated c-myc and MYCN gene amplification in three RMS cell lines (including FU-MMT-3) derived from three MMMTs by Southern blot analysis. FU-MMT-3 cells were propagated continuously for 57 serial passages over a 2-year period in vitro. FU-MMT-3 was able to produce tumours demonstrating pure RMS in athymic nude mice. Cytogenetically, FU-MMT-3 showed a triploidy pattern, with complex karyotypic abnormalities including trisomy of chromosome 8. All three RMS cell lines, including FU-MMT-3, showed amplification of the c-myc gene (approximately fourfold to eightfold), while no cell lines demonstrated MYCN gene amplification. FU-MMT-3 is considered to provide a useful system for the study of the biological behaviour of RMS in MMMTs. Extra copies of chromosome 8 and c-myc gene amplification may be associated with the rhabdomyoblastic differentiation in MMMT. Received: 7 January 1997 / Accepted: 2 May 1997     

Chromosomal location and structure of amplicons in two human cell lines with coamplification of c-myc and Ki-ras oncogenes

Gene amplification is a major mechanism through which oncogenes and genes responsible for drug resistance are overexpressed in neoplastic cells, and several models for structure of amplified units (amplicons) are postulated. In order to identify consistent changes associated with oncogene amplification, we analyzed chromosomal location and physical distance of amplicons of two independent human cell lines that have coamplified c-myc and Ki-ras oncogenes. In one cell line, KHC287, amplified c-myc genes were localized in two chromosomes and Ki-ras in three chromosomes. One marker chromosome was almost entirely encompassed by both amplified genes. In the other cell line, Lu-65, both of the amplified genes shared the same locus, on chromosome 12q+. The two genes, however, are more than 1500 kb apart in both cell lines. The above findings indicate that two different amplified genes became associated on one chromosome in two independent cell lines. This suggests that a common mechanism is associated with chromosomal rearrangements affecting different amplified genes.     

Differential expression of the c-kit proto-oncogene in germ cell tumours

The c-kit proto-oncogene product and its ligand stem cell factor play an important role in haematopoiesis, spermatogenesis, and melanogenesis. Using an anti-c-kit antiserum raised against a synthetic peptide, we studied the immunohistochemical expression of the c-kit gene product in 60 germ cell tumours (GCTs) (53 testicular, 7 extragonadal), derived from primary GCTs in 45 cases and metastatic tumours in 15 cases. Twenty-eight out of 28 seminomas showed c-kit membranous staining in the majority of cells. A similar pattern of expression was seen in intratubular germ cell neoplasia. Nine out of 29 (32 per cent) non-seminomas displayed cytoplasmic, but not membranous, c-kit immunoreactivity in occasional cells. In three mixed GCTs, c-kit expression was limited to the seminoma component. In normal testis, c-kit expression was observed in some basal tubular cells, corresponding to undifferentiated spermatogonia. These results suggest a role for c-kit in the oncogenesis of GCT, where down-regulation of c-kit might be a critical step during progression from seminomas to non-seminomas. Immunohistochemical analysis of c-kit should be considered as a diagnostic aid for GCT and in particular may be helpful in the identification of certain extragonadal seminomas.     

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.     

Hepatocyte growth factor stimulates invasion across reconstituted basement membranes by a new human small intestinal cell line

Hepatocyte growth factor/scatter factor (HGF/SF) is a protein growth factor whose pleiotropic effects on epithelial cells include the stimulation of motility, mitosis and tubulogenesis. These responses are mediated by the cell surface tyrosine kinase receptor c-met. Because both the cytokine and receptor are found in the gastrointestinal tract, we have studied the effects of HGF/SF on transformed gut epithelial cells which express c-met. Here we describe the response of a new transformed human jejunal epithelioid cell line (HIE-7) to HGF/SF. Morphologically HIE-7 cells are immature. Their epithelial lineage was confirmed by reactivity with the epithelial specific antibodies AE1/AE3, Cam 5.2, Ber-EP4 and anti-EMA and is consistent with their expression of c-met mRNA and protein. In addition, electron microscopic analysis revealed the presence of primitive junctions and rudimentary microvilli, but features of polarization were absent. When grown on reconstituted basement membranes, HIE-7 cells formed closely associated multicellular cord-like structures adjacent to acellular spaces. However, the cells did not mature structurally, form lumen-like structures or express disaccharidase mRNA, even in the presence of recombinant HGF (rHGF). On the other hand, rHGF induced HIE-7 cells to scatter and stimulated their rapid migration in a modified wound assay. To determine whether the motogenic effect caused by rHGF is associated with HIE-7 cell invasiveness across reconstituted basement membranes, a Boyden chamber chemoinvasion assay was performed. rHGF stimulated a 10-fold increase in the number of HIE-7 cells that crossed the basement membrane barrier, while only stimulating a small increase in chemotaxis across a collagen IV matrix, suggesting that the cytokine activates matrix penetration by these cells. rHGF also stimulated the invasion of basement membranes by an undifferentiated rat intestinal cell line (IEC-6) and by two human colon cancer cell lines which are poorly differentiated (DLD-1 and SW 948). In contrast, two moderately well differentiated colon cancer cell lines (Caco-2 and HT-29) did not manifest an invasive response when exposed to rHGF. These results suggest that HGF/SF may play a significant role in the invasive behavior of anaplastic and poorly differentiated gut epithelial tumors.Drs Sunitha and Meighen share first authorship of this paper.     

Expression of two keratins in basal cell,metatypical, and squamous cell carcinomas of the skin

Using an indirect immunoperoxidase method and monoclonal antibodies, the expression of keratins 8 and 17 is investigated in 53 cases of basal cell carcinoma, 10 cases of metatypical carcinoma, and 3 cases of squamous cell carcinoma. Basal cell carcinoma cells are found to express keratin 17 in all cases and keratin 8 in the majority of them. The levels of these keratins in metatypical carcinoma cells are much lower. In squamous cell carcinoma, the keratins are only expressed in a few fields of vision around areas of keratinization. Comparative microspectrophotometric measurements of the intensities of specific staining show that the histological preparations of basal cell carcinoma have optical densities 6–7 times higher than those of metatypical carcinoma and 10–14 times higher than those of squamous cell carcinoma. It is concluded that the features of keratin expression observed in this study may be of use in the differential diagnosis of skin cancers. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, N ^o 9, pp. 300–303, September, 1994 Presented by N. K. Permyakov, Member of the Russian Academy of Medical Sciences     

Transfection of Lymantria dispar insect cell lines

Lepidopteran cell lines derived from the gypsy moth, Lymantria dispar, have not been widely used in protein expression studies or systems because they are weakly adherent, have specific growth requirements and characteristics, and are generally difficult to transfect. Using lipid-mediated transfection of a reporter plasmid, we modify the standard method for transfection of L. dispar-derived embryonic cell lines IPLB-LdEp and -LdEIta, obtaining transfection efficiencies of 34% and 30%, respectively, as determined by image analysis assays. Using the standard lipid-mediated method, we obtain transfection efficiencies for L. dispar-derived cell line IPLB-Ld652Y of at least 40% with high mean expression levels, indicating the IPLB-Ld652Y cell line may be a superior choice for expression studies or systems requiring L. dispar-derived cells.     

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.     

N-Methyl-d-aspartate stimulation of the survival of rat cerebellar granule cells in culture is not dependent upon increased c-fos expression and is not mimicked by protein kinase C activation

The role of c-fos expression and protein kinase C in the survival of primary cultures of rat cerebellar granule cells was investigated. Results from immunocytochemistry and immunoblotting suggest that increased c-fos expression is not essential for the survival of cells grown in low K⁺ media in the presence of N-methyl-d-aspartate (NMDA) at the critical time point when sensitivity to survival requirements develops. In addition the phorbol ester, phorbol 12-myristate 13-acetate failed to bring about survival of cells cultured in low K⁺ media in the absence of NMDA when given chronically, suggesting that protein kinase C activation alone is not sufficient to maintain granule cell survival in culture.     

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 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     

Characterization and gene expression profiling in glioma cell lines with deletion of chromosome 19 before and after microcell‐mediated restoration of normal human chromosome 19

Nearly 10% of human gliomas are oligodendrogliomas. Deletion of chromosome arm 19q, often in conjunction with deletion of 1p, has been observed in 65–80% of these tumors. This has suggested the presence of a tumor suppressor gene located on the 19q arm. Chromosome 19 deletion is also of interest due to the better prognosis of patients with deletion, including longer survival and better response to chemotherapy, compared with patients without deletion. Two glioma cell lines with deletion of 19q were used for chromosome 19 microcell‐mediated transfer, to assess the effect of replacing the deleted segment. Complementation with chromosome 19 significantly reduced the growth rate of the hybrid cells compared with the parental cell lines. Affymetrix U133 Plus 2.0 Gene Chip analysis was performed to measure and compare the expression of the chromosome 19 genes in the chromosome 19 hybrid cell lines to the parental cell line. Probes were considered significantly different when a P value <0.01 was seen in all of the cell line comparisons. Of 345 probes within the commonly deleted 19q region, seven genes (APOE, RCN3, FLJ10781, SAE1, STRN4, CCDC8, and BCL2L12) were identified as potential candidate genes. RT‐PCR analysis of primary tumor specimens showed that several genes had significant differences when stratified by tumor morphology or deletion status. This suggests that one or more of these candidates may play a role in glioma formation or progression. © 2009 Wiley‐Liss, Inc.