Oncogene expression in endocrine pancreatie tumors

The mRNA expression of the (proto) oncogenes Ha-ras, Ki-ras, fos, c-myc, N-myc, and sis was studied in five pancreatic endocrine tumors and two non-neoplastic pancreases by in situ hybridization and Northern blot analysis. Ha-ras, Ki-ras, fos and c-myc, but not N-myc or sis mRNA was detected in all the tumors as well as in the non-neoplastic pancreatic tissues. Compared with non-tumorous pancreatic tissue, Ha-ras and Ki-ras mRNA was overexpressed up to 42-fold in all the tumors; metastasizing tumors showed 2–6 times higher Ha-ras mRNA levels than benign neoplasias. In contrast, c-myc mRNA levels were higher in normal tissue than in tumors and fos mRNA levels did not differ significantly between tumors and normal tissue. The activities of Ki-ras, fos and c-myc mRNA expression did not correlate with any of the histological or biological properties of the tumors, nor with the clinical course of disease. Our results, although based on a limited number of cases, suggest that Ha-ras and Ki-ras mRNA overexpression is associated with the development of pancreatic endocrine tumors. The measurement of Ha-ras mRNA levels may contribute to the assessment of tumor prognosis. Supported by grants of the “ Fonds zur F?rderung der Forschung” (P 5314) and by the “Jubil?umsfonds” of the Austrian National Bank (P 2889) to H.H.     

Oncogene expression in a medullary thyroid carcinoma

We report the expression of Ha-ras, fos, c-myc and N-myc mRNA in a human medullary carcinoma of the thyroid gland, both in primary tumor and lymph node metastasis, as demonstrated by in situ hybridization and Northern blot analysis. A significant difference in the oncogene expression in the primary tumor and the metastasis was not observed. Tumor tissue revealed a significant overexpression of Ha-ras, c-myc and N-myc mRNA as compared to the normal thyroid gland. The amount of fos mRNA expression in non tumorous thyroid gland did not significantly differ from tumor tissue, sis, fms and abl mRNA expression was not detectable in tumor tissue and non tumorous thyroid gland. We conclude, that the (over)expression of the oncogenes Ha-ras, c-myc and N-myc may be associated with initiation and progression of medullary thyroid carcinoma. Similar studies on additional cases of human medullary thyroid carcinoma will be necessary to reveal further information.     

Oncogene alterations in in vitro transformed rat tracheal epithelial cells.

10 derivations of rat tracheal epithelial (RTE) cells, including normal cells, normal primary cultures, 7 tumorigenic cell lines and 1 nontumorigenic cell line transformed in vitro by treatment with 7,12-dimethylbenz[a]anthracene (DMBA), benzo[a]pyrene (BP) and/or 12-O-tetradecanoylphorbol-13-acetate (TPA) were examined for oncogene alterations. No abnormalities of Ha-ras or Ki-ras were seen that were suggestive of amplification, rearrangement or the presence of RFLPs. Analysis of specific-point mutations in Ha-ras using Pst I digestion (codon 12, GGA to GCA) or Ha-ras and Ki-ras using Xba I (codon 61, CAA to CTA) were negative. In one cell line derived by DMBA treatment, changes in the c-myc restriction digest pattern were seen after incubation with Bam HI and Hind III. Northern analysis revealed consistent differences between normal and transformed cells when probed with Ha-ras; c-myc expression was of low intensity, and the expression of Ki-ras could not be detected. Transfection of RTE cell DNAs into NIH/3T3 cells did not result in the appearance of morphologic transformants. The studies suggest that Ha-ras or Ki-ras codon 61 A to T transversions (CAA to CTA) are not associated with the immortal/tumorigenic phenotype in RTE cells transformed by DMBA or TPA, and are in contrast to results reported in some other biological systems.     

The expression of oncogenes in human developing liver and hepatomas

Oncogene expression was examined in the human fetal liver and human hepatomas. Erb (B), erb (A+B), Ha-ras, myc, fos and fms oncogene expression elevated in certain stages of fetal liver development and in hepatoma as compared to the normal adult human liver. In contrast, rel, src, mos, sis, myb, Ki-ras and bas oncogenes showed no apparent change of their mRNA levels during fetal liver development and in hepatoma. Further study of erb B oncogene expression in human cirrhotic liver and hepatoma demonstrated a strong correlation between erb B expression and alteration of its gene structure.     

Differential regulation of the clusterin gene by Ha-ras and c-myc oncogenes and during apoptosis

Clusterin (ApoJ) is an extracellular glycoprotein expressed during processes of tissue differentiation and regression that involve programmed cell death (apoptosis). Increased clusterin expression has also been found in tumors, however, the mechanism underlying this induction is not known. Apoptotic processes in tumors could be responsible for clusterin gene activation. Alternatively, oncogenic mutations could modulate signal transduction, thereby inducing the gene. We examined the response of the rat clusterin gene to two oncogenes, Ha-ras and c-myc, in transfected Rat1 fibroblasts. While c-myc overexpression did not modify clusterin gene activity, the Ha-ras oncogene produced a seven to tenfold repression of clusterin mRNA; this down-regulation was also observed in the presence of c-myc. Since no induction of the clusterin gene was observed by the two oncogenes, we tested the alternative mechanism involving apoptosis. Growth factor withdrawal induced apoptosis, as shown by DNA degradation and micronuclei formation in the floating cells. Concomittantly we observed a three to tenfold increase in the amount of clusterin mRNA in the adhering cells of Rat1 and the c-myc transformed cell lines, and a weaker induction in the Ha-ras transformed cell line. On the basis of our results, we suggest that clusterin gene induction in the vital cells is produced by signaling molecules that are generated by the apoptotic cells. We conclude that apoptotic processes, not oncogenic mutations, are responsible for increased clusterin expression in tumors.     

CWE纯系小鼠脑组织发育过程中细胞癌基因及其表达研究

 本文以Ha-ras.c-fos、c-myc、v-erbB、mos五种癌基因片段为分子探针,应用斑点杂交技术,对CWE纯系小鼠脑组织发育的四个时期(胚胎期、新生期、性成熟期、体成熟期)的RNA进行分子杂交分析。发现:在实验各期中这几种癌基因的表达有较明显的变化。Ha-ras、c-fos、c-myc基因的表达在胚胎期和新生期脑组织中较高;v-erbB基因的表达在性成熟期脑组织中较高;mos基因在实验各期均无表达。DNA斑点杂交结果:小鼠脑组织发育各期均未观察到癌基因扩增现象。Southern杂交结果:CWE小鼠发育的各期均显示:5.4kb、3.4kb、1.8kb、1.0kb四条c-fos基因区带;7.4kb、4.5kb、3.4kb三条c-myc基因区带。结果表明:不同细胞癌基因在小鼠胚胎发育和胚后发育中有不同的表达。     

The expressions of oncogenes and liver-specific genes in Morris hepatomas

The expression of three liver-specific genes and four oncogenes was studied in the Morris hepatomas 8994, 7288c, 7777, 5123tc, and 7800. Total RNA isolated from these tumors was probed with cDNA's for alpha-fetoprotein (AFP), albumin, tyrosine aminotransferase (TAT), and the oncogenes Ha-ras, Ki-ras, myc and src. When compared to mRNA's levels expressed in normal adult liver, we found AFP levels elevated in AFP-producing tumors, albumin and TAT mRNA levels depressed in all tumors, except TAT is elevated in 5123tc and the oncogenes with the exception of src elevated in all tumors. These results argue against a coordinated expression of these genes as a result of transformation, but suggest that oncogene expression is related to tumorigenesis or proliferation.     

Rat embryo cells immortalized with transfected oncogenes are transformed by gamma irradiation.

Cesium-137 gamma rays were used to transform rat embryo cells (REC) which were first transfected with activated c-myc or c-Ha-ras oncogenes to produce immortal cell lines (REC:myc and REC:ras). When exposed to 6 Gy of 137Cs gamma rays, some cells became morphologically transformed with focus formation frequencies of approximately 3 x 10(-4) for REC:myc and approximately 1 x 10(-4) for REC:ras, respectively. Cells isolated from foci of gamma-ray-transformed REC:myc (REC:myc:gamma) formed anchorage-independent colonies and were tumorigenic in nude mice, but foci from gamma-ray-transformed REC:ras (REC:ras:gamma) did not exhibit either of these criteria of transformation. Similar to the results with gamma irradiation, we observed a sequence-dependent phenomenon when myc and ras were transfected into REC, one at a time. REC immortalized by ras transfection were not converted to a tumorigenic phenotype by secondary transfection with myc, but REC transfected with myc were very susceptible to transformation by subsequent ras transfection. This suggests that myc-immortalized cells are more permissive to transformation via secondary treatments. In sequentially transfected REC, myc expression was high whether it was transfected first or second, whereas ras expression was highest when the ras gene was transfected secondarily into myc-containing REC. Molecular analysis of REC:ras:gamma transformants showed no alterations in structure of the transfected ras or of the endogenous ras, myc, p53, or fos genes. The expression of ras and p53 was increased in some isolates of REC:ras:gamma, but myc and fos expression were not affected. Similarly, REC:myc:gamma transformants did not demonstrate rearrangement or amplification of the transfected or the endogenous myc genes, or of the potentially cooperating Ha-, Ki-, or N-ras genes. Northern hybridization analysis revealed increased expression of N-ras in two isolates, REC:myc:gamma 33 and gamma 41, but no alterations in the expression of myc, raf, Ha-ras, or Ki-ras genes in any REC:myc transformant. DNA from several transformed REC:myc:gamma cell lines induced focus formation in recipient C3H 10T1/2 and NIH 3T3 cells. The NIH 3T3 foci tested positive when hybridized to a probe for rat repetitive DNA. A detailed analysis of the NIH 3T3 transformants generated from REC:myc:gamma 33 and gamma 41 DNA failed to detect Ha-ras, Ki-ras, raf, neu, trk, abl, fms, or src oncogenes of rat origin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Consistent oncogene methylation changes in epithelial cells chemically transformed in vitro

We have examined the restriction digest patterns of CCGG sequences in Kiras, Ha-ras, and c-myc oncogenes in rat tracheal epithelial cells transformed in vitro by 7,12-dimethylbenz(a)anthracene, benzo(a)pyrene/12-O-tetradecanoylphorbol-13-acetate (TPA), or TPA alone. Oncogenes c-myc and Ha-ras in transformed cell lines, compared to normal rat tracheal epithelial cells and untreated primary cultures, had altered Hpa II restriction patterns as demonstrated by hybridizing bands of different molecular weight, or loss of bands. Ki-ras was hypermethylated in all cell derivations, including normal cells. These molecular alterations have not previously been reported for epithelial cells transformed in vitro by polycyclic hydrocarbons and tumor promoters, and suggest common mechanisms of action for agents with diverse molecular targets.     

Enhanced lytic susceptibility of Ha-ras transformants after oncogene induction is specific to activated NK cells

C3H 10T1/2 mouse fibroblasts were transfected with a plasmid vector composed of EJ, the mutated c-Ha-ras, and a metallothionein promotor that induced amplified ras expression when activated by culture in the presence of zinc. Experiments were conducted to compare the effect of induction on killing by activated natural killer (NK) cells, cytotoxic T lymphocytes, activated macrophages, and antibody plus complement. The only effector that recognized increased ras expression and exhibited high-inducible cytolysis was an activated NK cell. The effectors from spleen were poly I.C. boostable, Lyt-1.1 negative, NK 1.2 positive, and asialo GM1 positive. Spleen cells from T cell-deficient nude mice, but not NK-deficient beige mice, exhibited high levels of killing activity, and experiments with NK cell clones demonstrated that these lines were also highly cytolytic and killed Ha-ras transfectants in parallel to YAC. Transfection of the same fibroblast line with c-myc did not alter the level of activated NK sensitivity. Cold target competition experiments revealed that Ha-ras-transfected and non-transfected 10T1/2 fibroblasts competed equally for lysis of either YAC or Ha-ras transfectants. Rat-1 fibroblasts did not compete, but gained this capacity when transformed with the v-Ki-ras oncogene but not v-fps. These data suggest that Ha-ras acts in target cells at a post-binding step, whereas Ki-ras may affect expression of target-effector binding structures. The findings that activated NK cell lysis may be specifically influenced by ras expression support a role for NK cells in host surveillance against early neoplastic changes.     

CLDN11在胰腺癌神经浸润动物模型中mRNA水平的表达

目的:神经浸润的发生预示胰腺癌预后不良,疼痛的发生与神经浸润密切相关,癌细胞和神经组织间相互作用、连接及粘附可能参与了神经浸润的发生,Claudins作为组成紧密连接的主要成份,在多种肿瘤中有所表达,本实验拟通过观察其成员CLDN11在体内、体外mRNA水平的表达,探讨CLDN11在胰腺癌神经浸润发病机制中的作用,为其诊断及治疗新方法的探索提供一定的实验依据。方法:通过裸鼠坐骨神经周围注射不同人胰腺癌细胞系的方法建立稳定的胰腺癌神经浸润动物模型,成瘤后检测肿瘤组织中CLDN11 mRNA表达水平的差异。同时检测不同人胰腺癌细胞株中CLDN11 mRNA的表达水平的差异。结果:CLDN11在神经侵犯发生率低的肿瘤中的表达高于神经侵犯发生率高的肿瘤,在正常胰腺组织中无表达。CLDN11的mRNA水平在panc-1细胞株中表达高于Capan-2组。结论:经本实验研究发现CLDN11在PNI发生率高的肿瘤组织及高神经浸润能力的细胞株中表达下调,而在PNI发生率低的肿瘤组织及神经浸润能力低的细胞株中高表达,可以得出在神经浸润发生中,CLDN11的表达受到抑制的结论,由此推断如果过表达CLDN11,有可能阻碍PNI的发生及发展;另外,CLDN11表达的下降也可能预示着PNI的发生及进展,因此CLDN11表达的下降可作为PNI发生的预警信号,也可作为胰腺癌基因治疗的靶点,为提高胰腺癌的早期诊断率、改善胰腺癌的预后提供初步的基础实验依据。     

Haploinsufficient and predominant expression of multiple endocrine neoplasia type 1 (MEN1)-related genes, MLL, p27Kip1 and p18Ink4C in endocrine organs

Multiple endocrine neoplasia type 1 (MEN1) is a rare autosomal dominantly inherited syndrome characterized by parathyroid, gastro-entero-pancreatic and anterior pituitary tumors. Although the tissue selectivity of tumors in specific endocrine organs is the very essence of MEN1, the mechanisms underlying the tissue-selectivity of tumors remain unknown. The product of the Men1 gene, menin, and mixed lineage leukemia (MLL) have been found to cooperatively regulate p27(Kip1)/CDKN1B (p27) and p18(Ink4C)/CDKN2C (p18) genes. However, there are no reports on the tissue distribution of these MEN1-related genes. We investigated the expression of these genes in the endocrine and non-endocrine organs of wild-type, Men1 knockout and MLL knockout mice. Men1 mRNA was expressed at a similar level in endocrine and non-endocrine organs. However, MLL, p27 and p18 mRNAs were predominantly expressed in the endocrine organs. Notably, p27 and MLL mRNAs were expressed in the pituitary gland at levels approximately 12- and 17-fold higher than those in the liver. The heterozygotes of Men1 knockout mice the levels of MLL, p27 and p18 mRNAs did not differ from those in the wild-type mice. In contrast, heterozygotes of MLL knockout mice showed significant reductions in p27 mRNA as well as protein levels in the pituitary and p27 and p18 in the pancreatic islets, but not in the liver. This study demonstrated for the first time the predominant expression MEN1-related genes, particularly MLL and p27, in the endocrine organs, and a tissue-specific haploinsuffiency of MLL, but not menin, may lead to a decrease in levels of p27 and p18 mRNAs in endocrine organs. These findings may provide basic information for understanding the mechanisms of tissue selectivity of the tumorigenesis in patients with MEN1.     

骨桥蛋白在胰腺癌中的表达及其临床意义

目的:研究骨桥蛋白OPN在胰腺癌中的表达水平及其与临床病理特征的关系。方法:采用RT-PCR和免疫组织化学方法分别检测50例胰腺癌手术切除标本和20例癌旁正常胰腺组织中OPNmRNA及其蛋白水平的表达。结果:胰腺癌组织中OPNmRNA高表达率为90%(45/50)明显高于其在癌旁正常胰腺组织中的表达15%(3/20),差异有统计学意义(P<0.01);OPN蛋白的阳性率为86.0%(43/50),明显高于癌旁正常胰腺组织中的表达30%(6/20),差异有统计学意义(P<0.01);OPN在胰腺癌组织中表达与其淋巴结转移明显相关(P<0.05)。结论:OPN在胰腺癌中的过表达对胰腺癌的生长、浸润及转移起重要作用。     

Oncogene expression in vivo by ovarian adenocarcinomas and mixed-mullerian tumors

Six-micron paraffin sections of paraformaldehyde-fixed specimens of 24 ovarian benign and neoplastic specimens were assayed for tumor cell-specific oncogene expression by a sensitive, quantitative in situ hybridization technique with probes for 17 oncogenes, beta-actin, and E. coli beta-lactamase. In the benign, borderline, and invasive adenocarcinomas, multiple oncogenes, including neu, fes, fms, Ha-ras, trk, c-myc, fos, and PDGF-A chains, were expressed at significant levels relative to a housekeeping gene (beta-actin). In the mixed-Mullerian tumors, a rather different pattern of oncogene expression was observed, characterized primarily by expression of sis (PDGF-B chain). For the adenocarcinomas, statistical analysis demonstrated that expression of several genes (fms, neu, PDGF-A) was closely linked to others (c-fos, c-myc) known to have important roles in the control of cell proliferation, but only one gene, fms, correlated very strongly with clinicopathologic features (high FIGO histologic grade and high FIGO clinical stage) predictive of aggressive clinical behavior and poor outcome. The authors discuss the role that tumor epithelial cell expression of the fms gene product might play in the auto- and paracrine control of growth and dissemination of ovarian adenocarcinomas.