Metastatic phenotype of mouse-human melanoma cell hybrids is associated with the presence of chromosome 17 from highly metastatic human melanoma cells

We have previously shown that in interspecific mouse-human melanoma cell hybrids obtained by fusion of nonmetastatic mouse with metastatic human melanoma cells, the metastatic phenotype predominates. The purpose of this study was to identify human chromosome(s) which could be responsible for conferring metastatic potential upon nonmetastatic mouse melanoma cells and therefore harbor the genes important for the metastatic properties of human melanoma cells. Seven mouse-human melanoma hybrids were examined; five were derived from the fusion of nonmetastatic (C19) and metastatic (C3) mouse K-1735 melanoma clones with highly metastatic clone (C15) of human melanoma A375 and the two others had as a human partner a nonmetastatic clone (Cls) of the A375 melanoma. The hybrids were examined during segregation of human chromosomes in vitro and in vivo for metastatic properties in nude mice and for the retaining human chromosomes. In the hybrid H7, which demonstrated the highest metastatic potential, the presence of human chromosomes was studied by GTG-banding and by fluorescence in situ hybridization (FISH) analysis. In the other hybrids, only FISH detection of human chromosomes was applied. All hybrids derived from nonmetastatic mouse and metastatic human melanoma cells demonstrated metastatic properties from early passages, when they contained the majority of the human chromosomes. Their metastatic phenotype remained stable during further segregation of most of the human chromosomes except for 17. Chromosome 17 was retained most consistently in all examined hybrids. However, the metastatic phenotype of the hybrids was associated only with the presence of chromosome 17 from the metastatic human donor cells. This chromosome was also found in almost 100% of cells recovered from lung metastases derived from the hybrid cells. In one lung metastasis developed from the H7 hybrid, chromosome 17 was detected as the sole human chromosome and these cells preserved the acquired high metastatic properties. Based on these results we conclude that human chromosome 17 from metastatic melanoma cells (A375 C15), when functional in the mouse genetic background, can be sufficient to render the recipient nonmetastatic mouse cells to a fully malignant phenotype. Additional data suggest that this ability might be related to the expression of the mutated human p53 gene.     

Human chromosome 19 confers the CD2/E rosette receptor phenotype in interspecific cell hybrids.

Interspecific cell hybrids between Chinese Hamster Ovary (CHO) and phytohaemagglutinin (PHA) stimulated human T lymphocytes were purified by preparative rosetting with sheep red blood cells (SRBC). The hybrid cell clone used in the present study consisted of cells containing a complete set of the 20 CHO chromosomes and one extra human chromosome, No 19. Hybrid cells constitutively expressed high levels of human CD2 surface receptor and formed multilayer rosettes with SRBC and human erythrocytes. In addition to CD2 they produced low levels of a small number of human extracellular proteins. These findings suggest that the factor(s) responsible for CD2 expression are produced by the hybrid and that genes responsible for CD2 expression are located on chromosome 19. However, the present work cannot exclude that material of chromosome 1, where the CD2 gene has been assigned previously, is integrated somewhere in the hybrid karyotype. Further work is needed to clarify this point.     

Complement sensitivity of somatic hybrids of a complement-resistant murine leukemia cell line.

RADA-1 cells (H-2a), a murine leukemia cell line maintained by serial transfer in histocompatible recipients expressing thymus-leukemia (TL) 1, 2, 3 antigenic determinants, resisted the cytotoxic effects of guinea pig complement (GPC) and TL antiserum. The cells expressed a lower density of TL antigens than did ASL-1 cells, another TL(+) leukemia cell line expressing the same determinants and susceptible to complement (C)-mediated lysis. Stable somatic cell hybrids of RADA-1 cells and LM(TK)- cells (H2k), a TL(minus) thymidine kinase-deficient mutant of mouse L cells, were selected in hypoxanthine-aminopterin-thymidine medium. The hybrid cells expressed the H-2 antigens of both parents and shared a hybrid karyotype. They formed TL 1, 2, 3 antigens as determined by immunofluorescence, mixed hemagglutination methods, the direct isolation of TL antigens from Nonidet P40 extracts of the cells, and the capacity of the cells to reduce by absorption the known titers of TL antiserum. These hybrid cells lost the capacity to resist lysis by TL antiserum and GPC. They were susceptible to the cytotoxic effects of TL 1, 2, 3; TL 2; OR TL 1, 3 antiserum and GPC, even though the density of TL antigens associated with the cells was approximately 25% of their resistant RADA-1 parental cells. These results indicated that the mechanism of resistance to C-mediated lysis was genetically separable from the expression of TL antigens by the cells and that the susceptibility of the cells to the cytotoxic effects of antiserum was related only in part to the density of TL antigens expressed by the cells.     

The progression of gliomas is associated with cancer stem cell phenotype

Since cancer stem cells in brain tumors were introduced, there have been few explanations regarding the role of cancer stem cells in the progression of glioma. Here, we investigated their major molecular changes in tumor progression in relation to the stem cell subpopulation. Using 12 surgical specimens of gliomatosis cerebri (GC) in the early and advanced stages, we measured the expression of a panel of cell proliferation, microvessel density, microvessel areas, angiogenic factors and their associated receptors. In addition, expression of neural stem cell markers and associated cytokines were examined in tumor tissues by quantitative real-time RT-PCR. Comparing the biological characteristics between the initial infiltrating lesions (n=7) and progressed lesions (n=5), Sox2 and Musashi-1 were expressed in the tumor tissue at an early and a progressed state. Contrary to the early infiltrative phase representing angiogenesis-independent growth, GC with progression showed that nestin (+), PCNA (+) cells and total vessel area (angioectasia) were markedly increased with a higher expression of proangiogenic molecules and their receptors. These results suggest that tumor progression is mediated by cancer stem cells and cross-talk of cancer stem cells along with their environment and are closely associated with angiogenesis-dependent progression and -independent growth.     

Type I interferon resistance in a colorectal cancer cell line is associated with a more aggressive phenotype in vivo.

A type I interferon resistant variant (beta-MIP101) of the poorly differentiated human colon cancer cell MIP101 has a more aggressive phenotype in vivo in the nude mouse. Subcutaneous tumours grew at twice the rate of MIP101, but with similar morphology. beta-MIP101 also produced liver metastases at a higher frequency. beta-MIP101 tumours were diploid while MIP101 tumours were aneuploid. Both cell lines had doubling times of approximately 25 h in vitro.     

Suppression of tumorigenicity in somatic cell hybrids. II. Human chromosomes implicated as suppressors of tumorigenicity in hybrids with Chinese hamster ovary cells

Nontumorigenic diploid human cells were fused with tumorigenic Chinese hamster ovary cells (CHO), and the hybrids were tested for tumorigenicity to determine if specific human chromosomes are associated with suppression of tumorigenicity in cell hybrids. Chromosome complements of cells of 62 nontumorigenic and 45 tumorigenic hybrids (divided into those of low, medium, and high tumorigenicity) as well as 44 tumors derived from the tumorigenic hybrids were determined by both analysis of banded chromosomes and assays of gene markers. Although no single human chromosome was consistently associated with the suppressed phenotype, chromosome 2 was never found in tumor cells, and chromosomes 9, 10, 11, and 17 were found at very low incidences in tumor cells, which suggested that they carry tumorigenicity suppressor information. Since not all suppressed hybrids contained these chromosomes, it is likely that they suppressed tumorigenicity only in combination with each other or other chromosomes. Nine chromosomes in 12 pairwise combinations of nonhomologous chromosomes were not found in tumor cells and were found at an incidence of 5% or less in hybrids of both medium and high tumorigenicity. Other experiments implicated 11 of these combinations involving only 8 chromosomes (chromosomes 4, 7, 8, 9, 10, 11, 13, and 17) as those primarily involved in suppression. Whether chromosome 2 requires another chromosome to effect suppression could not be determined. Further evaluations of the implicated suppressors, including selection of tumorigenic segregants from a panel of suppressed hybrids, again implicated the same chromosomes and their combinations in suppression. Oncogenes have been mapped to many of these chromosomes, and they are frequently involved in tumor-type-specific numerical or structural abnormalities in human neoplasias. The combined evidence suggests that specific human chromosomes of a normal cell carry genes that can regulate several cell phenotypes necessary for the expression of tumorigenicity.     

Modification of myc gene amplification in human somatic cell hybrids

In order to study whether cell fusion would modify the DNA copy number of an amplified oncogene, somatic cell hybrids were made between the human neuroepithelioma cell line MCIXC and HeLaCOT human adenocarcinoma cells. MCIXC contains approximately 21 copies of the c-myc oncogene and HeLaCOT contains approximately 5 copies relative to the control. All hybrid clones investigated displayed a marked decrease in the number of copies of c-myc DNA (an average of 5 copies), while the level of c-myc RNA in the hybrids was similar to that found in both parents. All eight hybrid clones were found to be completely nontumorigenic even though both parent cells formed tumors in 100% of the nude mice treated by injection. This loss of oncogene amplification in the hybrids was shown not to be due to either heterogeneity of c-myc amplification in the MCIXC parent or segregation of a copy of the chromosome 22 from the hybrids. This loss most likely resulted from the breakdown of a homogeneously staining region (containing the amplified gene copies) into double minutes, which were subsequently lost from the cells. The HeLaCOT cell line was also fused to the human neuroblastoma BE(2)C, which contains approximately 123 copies of the N-myc oncogene relative to control. Ten hybrid clones were found to contain an average of 47 copies of N-myc DNA, significantly less than the 91 copies predicted had no loss occurred. These BE(2)C x HeLaCOT hybrids expressed on average about 15% the N-myc RNA seen in the BE(2)C parent and, as with the MCIXC x HeLaCOT hybrids, were found to be completely nontumorigenic. However, upon passage in culture, one BE(2)C x HeLaCOT hybrid eventually became tumorigenic. This hybrid also displayed reduced copies of N-myc DNA in comparison to its parent hybrid but surprisingly showed a 2-fold increase in N-myc RNA. Thus, the expression of N-myc, but not the amplification state of either myc gene, appears to correlate with the tumorigenicity of the cells.     

Decreased adiponectin level is associated with aggressive phenotype of tongue squamous cell carcinoma

Circulating adiponectin levels are inversely associated with risk of various obesity‐related cancers. However, the effect of adiponectin on carcinogenesis and progression of tongue squamous cell carcinoma (TSCC) remains unknown. We measured serum adiponectin levels in 59 patients with TSCC and 50 healthy controls. Expression of adiponectin and its receptors in paired tumor and paracancerous specimens were determined by immunohistochemical staining (n = 37) and western blot (n = 30), respectively. Serum adiponectin level was lower in patients than in controls (5.0 ± 2.4 vs 8.4 ± 3.5 μg/mL, P < 0.01), and was inversely associated with histological grade and lymph node metastasis but not tumor size. Local adiponectin levels in tumor tissue gradually decreased as tumor‐node‐metastasis stage increased, while the expression of adiponectin receptors was unchanged. In addition, serum adiponectin levels in the TSCC patients without metabolic and cardiovascular diseases, or without smoking and drinking habits, were still lower than in controls. Furthermore, adiponectin inhibited the migration, but not proliferation, of SCC15 cells in vitro. These results indicate that a decreased adiponectin level is associated with risk of TSCC. Hypoadiponectinemia might be used as a biomarker to predict an aggressive phenotype of TSCC.     

Intrinsic gemcitabine resistance in a novel pancreatic cancer cell line is associated with cancer stem cell-like phenotype

Pancreatic ductal adenocarcinoma (PDA) remains one of the most lethal malignancies in the world, often diagnosed at an advanced stage, resistant to conventional chemotherapy and having high invasive and metastatic potential. The mechanism of drug resistance of PDA is still not clear. In the present study, we established two novel pancreatic cancer cell lines PAXC-002 and PAXC-003 from human primary xenograft models. The cell lines were characterized by morphology, karyotype, pancreatic cancer marker and short tandem repeat (STR) analysis, and growth kinetics and tumorigenicity. The in vitro anti-proliferation test revealed that PAXC-002 cell was intrinsically resistant to the standard of care chemotherapy-gemcitabine, compared with that of PAXC-003 and other widely used pancreatic cancer cell lines. Interestingly, the gemcitabine resistant PAXC-002 cell line was more potent in forming colonies in 3-Dimensional matrigel culture conditions and had a higher percentage of CD133 positive cells, which is recognized as a cancer stem cell marker, compared to the gemcitabine-sensitive PAXC-003 cell line. In this study, we present two novel pancreatic cancer cell lines which could be used for gemcitabine resistance investigation, mechanism identification of pancreatic cancer and anticancer drug screening. The preliminary data indicate that the drug resistance of pancreatic carcinoma cells is associated with a cancer stem cell-like phenotype.     

Characterization of human keratinocyte X HeLa somatic cell hybrids

Intraspecific human cell hybrids were created by fusing normal epidermal keratinocytes with carcinoma (HeLa) cells. All of the hybrids were epithelial in morphology and exhibited a bright cytoskeletal pattern after indirect immunofluorescent labelling by antibody against keratin. Like the normal parental cells, the hybrid populations had organized arrays of microfilaments, and expressed low levels of surface fibronectin, predominantly in short "stitches" at cell boundaries. None of the cells expressed collagen type I, as expected of epithelial cells. Subcutaneous injection into nude mice revealed that the tumorigenic phenotype was initially suppressed in certain of the hybrids. However, cells of these lineages tested at later population doublings, and other hybrid clones tested at early population doublings, formed very small, non-progressive nodules, Histologically, these nodules resembled moderately to well-differentiated squamous-cell carcinomas. The properties in vitro and in vivo of these epithelial hybrids are compared to those of human fibroblast X HeLa hybrids.     

Repression of an alternative mechanism for lengthening of telomeres in somatic cell hybrids.

Some immortalized cell lines maintain their telomeres in the absence of detectable telomerase activity by an alternative (ALT) mechanism. To study how telomere maintenance is controlled in ALT cells, we have fused an ALT cell line GM847 (SV40 immortalized human skin fibroblasts) with normal fibroblasts or with telomerase positive immortal human cell lines and have examined their proliferative potential and telomere dynamics. The telomeres in ALT cells are characteristically very heterogeneous in length, ranging from very short to very long. The ALT x normal hybrids underwent a rapid reduction in telomeric DNA and entered a senescence-like state. Immortal segregants rapidly reverted to the ALT telomere phenotype. Fusion of ALT cells to telomerase-positive immortal cells in the same immortalization complementation group resulted in hybrids that appeared immortal and also exhibited repression of the ALT telomere phenotype. In these hybrids, which were all telomerase-positive, we observed an initial rapid loss of most long telomeres, followed either by gradual loss of the remaining long telomeres at a rate similar to the rate of telomere shortening in normal telomerase-negative cells, or by maintenance of shortened telomeres. These data indicate the existence of a mechanism of rapid telomere deletion in human cells. They also demonstrate that normal cells and at least some telomerase-positive immortal cells contain repressors of the ALT telomere phenotype.     

Elevated Bcr-Abl expression levels are sufficient for a haematopoietic cell line to acquire a drug-resistant phenotype.

A characteristic feature of chronic myeloid leukaemia (CML) is the inevitable advancement from a treatable chronic phase to a fatal, drug-resistant stage referred to as blast crisis. The molecular mechanisms responsible for this disease transition remain unknown. As increased expression of Bcr-Abl has been associated with blast crisis CML, we have established transfectants in 32D cells that express low and high levels of Bcr-Abl, and assessed their drug sensitivity. Cells with high Bcr-Abl expression levels are resistant to conventional cytotoxic drugs, and also require higher levels of STI571 (an inhibitor of Bcr-Abl), to induce cell death. Co-treatment with cytotoxic drugs and STI571 increased the sensitivity of the drug-resistant cells. Despite the drug-resistant phenotype, high Bcr-Abl levels concomitantly increased the expression of p53, p21, Bax and down-regulated Bcl-2. These cells maintain a survival advantage irrespective of a reduced proportion of cycling cells and the pro-apoptotic shift in gene expression. In addition, the level of Bcr-Abl expression (high or low) does not alter the growth factor independence and elevated Bcl-xL expression observed. Our study indicates that drug resistance can be primarily attained by increased Bcr-Abl expression, and highlights the potential of therapy which combines STI571 with conventional cytotoxic drugs.     

Overexpression of the p16 cell cycle inhibitor in breast cancer is associated with a more malignant phenotype

In order to study the role of the p16^INK4A(MTS1/CDKN2a) tumor suppressor in breast cancer, we analyzed p16 protein expression in 60 breast cancer samples which were also analyzed for expression of Rb, Ki67, HER2/neu, and estrogen and progesterone receptors (ER, PR). P16 expression was investigated by two methods: western blotting (WB) followed by densitometry, and immunohistochemistry (IHC). The Rb status was studied by western blotting, and expression of Ki67, HER2/neu, ER, and PR was analyzed immunohistochemically. P16-negative results were found in 18% of the carcinomas by WB, but in only one case by IHC and were not associated with established prognostic parameters. In contrast, p16 overexpression which was detected by WB and IHC in 15% and 25% of the tumors, respectively, was significantly associated with unfavorable prognostic indicators. High p16 expression as detected by both methods correlated significantly with high grading and a negative estrogen receptor status. In addition, a significant association of p16 staining with inverse progesterone receptor status and high Ki67 expression was found with IHC. No correlation of p16 expression with clinical stage, HER2/neu immunostaining, Rb expression or Rb phosphorylation was found. Comparison of western blot results and immunohistochemistry suggests that both nuclear and cytoplasmic immunoreactivity in tumor cells is specific and due to p16 expression. We conclude that high p16 reactivity (both nuclear and cytoplasmic) is indicative of a more undifferentiated, malignant phenotype in mammary carcinomas.     

Characteristics of cell membranes from somatic cell hybrids between rat hepatoma and mouse L-cells.

Cell membranes from mouse L-cells (L-B82), rat hepatoma (HTC-H1), and three clones of their somatic cell hybrids (07, V4a, and V5) showing different degrees of density-dependent inhibition of growth were analyzed by polyacrylamide gel electrophoresis. The membrane polypeptides of the hybrid clones were all similar and all showed higher proportions of polypeptides with molecular weights of 56,000 and 45,000 than their parents of their normal counterparts. The major glycoprotein form cell hybrids appeared to be identical with that of rat liver or rat hepatoma cells and different from that of L-cells. One hybrid showed density-dependent inhibition growth; the other two, like both parents, did not. All produced tumors in nude mice, although tumor production by the hybrids was delayed. A large external protein (M.W. 240,000) iodinated by lactoperoxidase-catalyzed reaction was virtually missing in the parents but was present at high levels in all their hybrid clones. Thus, there was a lack of correlation between the presence of this protein, growth control in vitro, and tumorigenicity. Furthermore, no correlation was seen between agglutination of these cells by concanavalin A and tumorigenicity. The factors controlling these membrane properties thus are independent of density-dependent inhibition of growth and of those controlling the expression of cancer.     

Sirt7在宫颈鳞状细胞癌组织中的表达及意义

目的:作为七个Sirtuin(SIRT,又称抗衰老酶)家族成员(Sirt1-Sirt7)之一,Sirt7已被发现在很多肿瘤中异常表达,但其在宫颈癌中的表达仍不清楚.本研究中重点探索Sirt7在宫颈鳞状细胞癌中的表达以及其在宫颈癌发生、发展及转移方面的临床意义.方法:本研究共纳入手术切除宫颈癌组织石蜡标本113例,采用免疫组化(SP)法检测宫颈癌标本39例,宫颈上皮内瘤变标本62例和正常宫颈组织12 例中Sirt7的表达情况,并分析Sirt7与宫颈癌临床病理之间的关系.结果:Sirt7在正常宫颈、宫颈鳞状上皮内瘤变(低度)、宫颈鳞状上皮内瘤变(高度)及宫颈鳞癌组织中的阳性表达率依次上升,分别为 8.3%,13.1%,41.7% 及69.2%(P<0.05).另外,Sirt7的表达情况与宫颈鳞癌的组织分化程度相关(P<0.05),具有统计学意义,而与临床分期、淋巴结转移及患者年龄无关(P>0.05).结论:Sirt7可能在宫颈鳞癌的恶性进展中发挥作用,有可能是宫颈鳞癌治疗的一个潜在靶点.