Oncogene amplification and chromosomal abnormalities in small cell lung cancer

Twelve cell lines isolated from patients with small cell lung cancer have been studied for amplification of the three characterised members of the myc proto-oncogene family (c-myc, N-myc, and L-myc) and for abnormalities of chromosome 3. Ten of these lines were being studied for the first time. Ten of the 12 small cell lung cancer cell lines had amplification of one member of the myc proto-oncogene family. Amplification of c-myc was observed in only one small cell lung line--a "morphological variant". One "classic" small cell lung cancer line expressed c-myc but had no obvious amplification of the gene. N-myc and L-myc were more commonly amplified than c-myc. Chromosomal abnormalities (mainly deletions) in chromosome 3 were observed in all small cell lung carcinoma cell lines examined. When the small cell lung carcinoma lines were grouped according to "classic" or "variant" characteristics, it was found that the "classics" had deletions of the short arm of chromosome 3, whereas the "biochemical variants" had deletions of the long arm of chromosome 3. The extent of the deletions varied between cell lines. For the deletion in the short arm of chromosome 3 the minimum common region of overlap was assigned to bands 3p23-3p24.     

人脑原发性肿瘤癌基因扩增和重排的研究

作者对52例人脑原发性肿瘤和5例正常人脑DNA中c-myc、L-myc、Nmyc、erbB、c-fos、sis及Ha-ras等七种癌基因的扩增和重排进行了研究,发现多数胶质瘤中有c-myc、L-myc、erbB及c-fos等癌基因的扩增,少数胶质瘤和脑膜瘤中发现myc家族癌基因的限制性酶切区带位置有多态性变化;同时还观察到原发性脑瘤中存在两种或两种以上癌基因的扩增和重排现象。作者对癌基因与人脑原发性肿瘤的关系进行了讨论。     

Oncogene amplification correlates with dense lymphocyte infiltration in human breast cancers: A role for hematopoietic growth factor release by tumor cells?

One hundred six primary breast cancer samples were analysed for c-erbB2, int-2, and c-myc gene amplification. Surgically confirmed nodal involvement was observed in 42%. Level of gene amplification was studied by Southern and/or slot blot techniques. Amplified c-erbB2 gene sequences were present in 21.5% of all samples. Int-2 was amplified in 13.1% and c-myc was amplified in 10.3%. In a non-parametric test (Kruskal-Wallis) a strong negative association was found between high levels of c-erbB2 amplification and absence of estrogen receptor (ER) (P = .0009) or progesterone receptor (PR) (P = .011) expression. No correlations were found between all or high levels of amplification of each oncogene separately or combined with T, N, grade, multifocality of tumor, or associated carcinoma in situ. There was a trend approaching statistical significance for patients with c-erbB2 amplifications to have positive lymph nodes at surgery (P = 0.09). A somewhat surprising finding however was a very strong association between oncogene amplification and dense lymphocyte infiltration of the tumor (P = .05). This correlation is even stronger when only high levels of amplification are considered, either for each oncogene separately (P = .0048) or in combination (P = .0007). We propose that malignant cell cytokine production may help explain this observation.     

Rapid phosphorylation of the L-myc protein induced by phorbol ester tumor promoters and serum.

We have examined post-translational modification of the L-myc protein using polyclonal and monoclonal antibodies against a peptide well conserved in the predicted amino acid sequences of the c-myc, N-myc and L-myc genes. These antibodies precipitate three polypeptides of Mr 60-66,000 from [35S]methionine or [32P]orthophosphate-labelled human small cell lung cancer cell lines expressing amplified L-myc genes, but not the other myc genes. Treatment of the L-myc immunoprecipitates with alkaline phosphatase prior to electrophoresis converts the three methionine-labelled polypeptides into a single band migrating at Mr 59,000, and efficiently removes radioactivity from the 32P-labelled L-myc protein, suggesting that, in contrast to the c-myc and N-myc proteins, the L-myc polypeptide heterogeneity is due to differential phosphorylation of a common precursor. When the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or serum is added to cultures of U-1690 cells the Mr 66,000 polypeptide is rapidly enriched while the Mr 60,000 form is decreased in the L-myc immunoprecipitates. This effect is correlated with the ability of phorbol ester and diacylglycerol analogues to activate protein kinase C. The TPA-induced phosphorylation of the L-myc protein occurs in a protein synthesis-independent manner as it is not inhibited by cycloheximide or anisomycin. These data indicate that the phosphorylation of the L-myc nuclear oncoprotein is modulated in response to TPA via a rapid signal transduction system involving protein kinase C. This mechanism could play an important role in the response of lung cells to e.g. bombesin-related growth factors.     

A novel combination of K-ras and myc amplification accompanied by point mutational activation of K-ras in a human lung cancer.

Amplifications of two oncogenes, c-K-ras-2 and c-myc, were found in a human lung giant cell carcinoma (LGCC) Lu-65, which is maintained in nude mice. The extent of c-K-ras-2 and myc amplifications were estimated to be 10- and 8-fold, respectively, by means of the Southern hybridization procedure. In addition, NIH3T3 cells were transformed by transfection of Lu-65 DNA and the transforming gene was identified as c-K-ras-2. c-K-ras-2 genes were cloned from a gene library of Lu-65 and a single point mutation causing a substitution of cysteine for glycine in codon 12 was found by DNA sequencing. It was concluded that the amplification of the c-myc and c-K-ras-2 genes are accompanied by point mutational activation of c-K-ras-2 in the human LGCC Lu-65. This is the first report of multiple gene amplification accompanied by a point mutation of oncogenes in human cancer cells, providing further support for the idea that co-operation of at least two activated cellular oncogenes is required for carcinogenesis.     

Promotion of growth and apoptosis in c-myc nullizygous fibroblasts by other members of the myc oncoprotein family

c-myc nullizygous fibroblasts (KO cells) were used to compare the abilities of c-myc, N-myc and L-myc oncoproteins to accelerate growth, promote apoptosis, revert morphology, and regulate the expression of previously described c-myc target genes. All three myc oncoproteins were expressed following retroviral transduction of KO cells. The proteins all enhanced the growth rate of KO cells and significantly shortened the cell cycle transition time. They also accelerated apoptosis following serum deprivation, reverted the abnormal KO cell morphology, and modulated the expression of previously described c-myc target genes. In most cases, L-myc was equivalent to c-myc and N-myc in restoring all of the c-myc-dependent activities. These findings contrast with the previously reported weak transforming and transactivating properties of L-myc. Myc oncoproteins may thus impart both highly similar as well as dissimilar signals to the cells in which they are expressed.     

L-myc cooperates with ras to transform primary rat embryo fibroblasts.

Recent molecular analysis has revealed that L-myc has several domains of extremely conserved amino acid sequence homology with c-myc and N-myc, suggesting similarity of function. We tested the biologic activity of L-myc by using an expression vector containing a cDNA clone coding for the major open reading frame in the 3.9-kilobase mRNA of L-myc under the control of a strong promoter (Moloney long terminal repeat) and found that L-myc complemented an activated ras gene in transforming primary rat embryo fibroblasts. However, the efficiency of transformation was 1 to 10% of that seen with the c-myc and simian virus 40 (SV40) controls. The L-myc/ras transformants initially grew more slowly than c-myc or SV40 transformants, but once established as continuous cell lines, they were indistinguishable from cell lines derived from c-myc/ras or SV40/ras transfectants as determined by morphology, soft-agar cloning, and tumorigenicity in nude mice.     

Contrasting expression patterns of three members of the myc family of protooncogenes in the developing and adult mouse kidney

The myc family of protooncogenes encode similar but distinct nuclear proteins. Since N-myc, c-myc, and L-myc have been found to be expressed in the newborn kidney, we studied their expression during murine kidney development. By organ culture studies and in situ hybridization of tissue sections, we found that each of the three members of the myc gene family shows a remarkably distinct expression pattern during kidney development. It is known that mesenchymal stem cells of the embryonic kidney convert into epithelium if properly induced. We demonstrate the N-myc expression increases during the first 24 h of in vitro culture as an early response to induction. Moreover, the upregulation was transient and expression levels were already low during the first stages of overt epithelial cell polarization. In contrast, neither c-myc nor L-myc were upregulated by induction of epithelial differentiation. c-myc was expressed in the uninduced mesenchyme but subsequently became restricted to the newly formed epithelium and was not expressed in the surrounding loose mesenchyme. At onset of terminal differentiation c-myc expression was turned off also from the epithelial tubules. We conclude that N-myc is a marker for induction and early epithelial differentiation states. That the undifferentiated mesenchyme, unlike stromal cells of later developmental stages, express c-myc demonstrates that the undifferentiated mesenchymal stem cells are distinct from the stromal cells. The most astonishing finding, however, was the high level of L-myc mRNA in the ureter, ureter-derived renal pelvis, papilla, and collecting ducts. In the ureter, expression increased, rather than decreased, with advancing maturation and was highest in adult tissue. Our results suggest that each of the three members of the myc gene family are involved in quite disparate differentiation processes, even within one tissue.     

Amplified cellular oncogenes in neoplasms of the human central nervous system.

Significant advances have recently been made in a number of areas concerning central nervous system (CNS) neoplasia. Particularly salient are the following: (1) gene amplification is related to increasing grade of human glioma malignancy and occurs in approximately 40% of the most common and most malignant variety of glioma, glioblastoma multiforme (GBM), (2) by far the most commonly amplified gene in glioblastomas is the epidermal growth factor receptor (EGFR) gene, which is amplified in about one third of GBMs, (3) a small percentage of GBMs amplify N-myc or the novel sequence gli, (4) the EGFR gene is rearranged in at least half of gliomas in which it is amplified, and (5) EGFR gene rearrangement results in external domain deletions that yield truncated EGF receptors. Antibodies specific for the mutant EGF receptor fusion junction have been successfully produced and provide stimulating new potential avenues for tumor imaging and therapy. For pediatric CNS neoplasms, only medulloblastoma has been investigated in adequate numbers; a small percentage exhibit amplification of either the N-myc or c-myc genes.     

Contrasting roles for c-Myc and L-Myc in the regulation of cellular growth and differentiation in vivo.

Although myc family genes are differentially expressed during development, their expression frequently overlaps, suggesting that they may serve both distinct and common biological functions. In addition, alterations in their expression occur at major developmental transitions in many cell lineages. For example, during mouse lens maturation, the growth arrest and differentiation of epithelial cells into lens fiber cells is associated with a decrease in L- and c-myc expression and a reciprocal rise in N-myc levels. To determine whether the down-regulation of L- and c-myc are required for mitotic arrest and/or completion of differentiation and whether these genes have distinct or similar activities in the same cell type, we have studied the consequences of forced L- and c-myc expression in the lens fiber cell compartment using the alpha A-crystallin promoter in transgenic mice (alpha A/L-myc and alpha A/c-myc mice). With respect to morphological and molecular differentiation, alpha A/L-myc lenses were characterized by a severely disorganized lens fiber cell compartment and a significant decrease in the expression of a late-stage differentiation marker (MIP26); in contrast, differentiation appeared to be unaffected in alpha A/c-myc mice. Furthermore, an analysis of proliferation indicated that while alpha A/L-myc fiber cells withdrew properly from the cell cycle, inappropriate cell cycle progression occurred in the lens fiber cell compartment of alpha A/c-myc mice. These observations indicate that continued late-stage expression of L-myc affected differentiation processes directly, rather than indirectly through deregulated growth control, whereas constitutive c-myc expression inhibited proliferative arrest, but did not appear to disturb differentiation. As a direct corollary, our data indicate that L-Myc and c-Myc are involved in distinct physiological processes in the same cell type.     

Integration of papillomavirus DNA near myc genes in genital carcinomas and its consequences for proto-oncogene expression.

DNA sequences of specific human papillomavirus (HPV) types are found integrated in the cell genome in most invasive genital carcinomas. We have determined the chromosomal localization of integrated HPV type 16 (HPV-16) or HPV-18 genomes in genital cancers by in situ hybridization experiments. In three cancers, HPV sequences were localized in chromosome band 8q24.1, in which the c-myc gene is mapped, and in one cancer HPV sequences were localized in chromosome band 2p24, which contains the N-myc gene. In three of the four cases, the proto-oncogene located near integrated viral sequences was found to be structurally altered and/or overexpressed. These data indicate that HPV genomes are preferentially integrated near myc genes in invasive genital cancers and support the hypothesis that integration plays a part in tumor progression via an activation of cellular oncogenes.     

Amplified DNA sequences in cancers

Amplification of genes other than known oncogenes was analyzed using an in-gel DNA renaturation method, in which a mixture of restriction fragments of radioactively labelled tracer DNA and unlabelled driver DNA was electrophoresed and amplified DNA fragments were visualized after two cycles of denaturation and renaturation in the gel. Different DNA fragments were found to be amplified more than 400 fold in NB1, a neuroblastoma cell line, in Y79, a retinoblastoma cell line and in H69, a small cell lung carcinoma cell line, in addition to 120 to 160-fold amplification of N-myc gene in these three cell lines.