Alterations In Metaphase Durations In Cells Derived From Human Tumours

With the use of time-lapse cinemicrography, we previously found that metaphase durations were significantly prolonged in SV40-transformed human fibroblasts when compared to untransformed controls. This was consistent with some earlier reports and suggested that prolonged metaphases could account for high metaphase/prophase ratios and possibly, in part, for increased mitotic indices seen in advanced tumours. However, there are inconsistencies in the literature and no comparable data available from malignant carcinomas. Presented in this paper are data from two cervical dysplasias, two cases of carcinoma in situ, nine malignant carcinomas and several other types of human cells. the results show that mean metaphase durations were prolonged in cells derived from most of the carcinomas but not from the other cell types. On the other hand, cytokinesis appears to progress more rapidly than normal in most of the tumour-derived cells. These and other findings indicate that the changes are a result of some metabolic alteration common to many but not all tumour cells. For reasons presented, we suggest as a working hypothesis that the alterations may be due to changes in calcium regulation, possibly resulting from alterations in mitochondrial metabolism.     

Clonal growth of normal human epidermal keratinocytes in a defined medium

Early studies on the duration of mitotic stages and on metaphase-to-prophase ratios in a number of normal and neoplastic cells indicated that the process of mitosis becomes altered during the course of oncogenesis. However, the nature of these changes and their effects on each of the mitotic stages are still unclear. With the use of time lapse cinemicrography, we have compared the durations of mitotic stages of SV40/Wl-38 and SV40/Wl-26 cells to those of their normal counterparts and to other nontransformed human fibroblasts. We also examined the relative frequencies of the individual mitotic stages in fixed preparations of Wl-38 and SV40/Wl-38 cells. The data show that metaphase durations are increased in the transformed cells and as much as 3-4.7-fold in SV40/Wl-38 cells compared to Wl-38 and other nontransformed cells. Other stages are also prolonged though to lesser degrees. These findings suggest that increased metaphase/prophase ratios observed in many tumors are due to increases in duration of metaphase rather than to shorter prophases, and that increased mitotic indices commonly observed in malignant tumors and sometimes used as an index of growth rate are at least in part due to the prolongation of mitotic stages.     

Expression of adhesion molecules and mucins in human and rhesus macaque gastrointestinal epithelial cells

Epithelial junctions and mucins play key roles in the gastrointestinal mucosal barrier, and their alterations are associated with numerous diseases, including carcinomas. The systematic expression of adhesion molecules and mucins in normal and malignant human gastrointestinal cells was investigated in this study. In normal human gastrointestinal cells, zonula occludens-1 (ZO-1), α-catenin, β-catenin, γ-catenin and desmoglein-2 (DSG2) were located in the cytoplasmic membranes, whereas symplekin stained in the nuclei. ZO-1, the three catenins, and DSG2 were observed in the gastric and colorectal carcinomas with reduced and heterogeneous expression and with abnormal distribution. Symplekin was detected in the nuclei of tumor cells in most tumors but not observed in some others. The immunohistochemical results for ZO-1 and symplekin on the tissues were consistent with the data for the cultured cells obtained by immunocytochemical staining and Western blot analysis. MUC1 was not stained in the normal gastrointestinal cells without periodate oxidation, but it was strongly labeled in the malignant gastrointestinal cells. MUC2 was detected in the normal and malignant gastrointestinal cells without the periodate treatment. These findings indicate that alterations in the expression of the epithelial junctions and mucins are associated with the malignant transformation of gastrointestinal cells. In addition, the gastrointestinal epithelial cells of rhesus macaques expressed these adhesion molecules and mucins, as did the human cells, suggesting that the rhesus monkey is a suitable experimental animal model for research on adhesion molecules and mucins.     

Genetic alterations in poorly differentiated and undifferentiated thyroid carcinomas

Thyroid gland presents a wide spectrum of tumours derived from follicular cells that range from well differentiated, papillary and follicular carcinoma (PTC and FTC, respectively), usually carrying a good prognosis, to the clinically aggressive, poorly differentiated (PDTC) and undifferentiated thyroid carcinoma (UTC).It is usually accepted that PDTC and UTC occur either de novo or progress from a pre-existing well differentiated carcinoma through a multistep process of genetic and epigenetic changes that lead to clonal expansion and neoplastic development. Mutations and epigenetic alterations in PDTC and UTC are far from being totally clarified. Assuming that PDTC and UTC may derive from well differentiated thyroid carcinomas (WDTC), it is expected that some PDTC and UTC would harbour genetic alterations that are typical of PTC and FTC. This is the case for some molecular markers (BRAF and NRAS) that are present in WDTC, PDTC and UTC. Other genes, namely P53, are almost exclusively detected in less differentiated and undifferentiated thyroid tumours, supporting a diagnosis of PDTC or, much more often, UTC. Thyroid-specific rearrangements RET/PTC and PAX8/PPARγ, on the other hand, are rarely found in PDTC and UTC, suggesting that these genetic alterations do not predispose cells to dedifferentiation. In the present review we have summarized the molecular changes associated with the two most aggressive types of thyroid cancer.     

Bimolane induces multiple types of chromosomal aberrations in human lymphocytes in vitro

Bimolane has been commonly used in China for the treatment of psoriasis and various types of cancer. Patients treated with bimolane have been reported to have an increased risk of developing therapy-related leukemias. Although bimolane has been identified as a human leukemia-inducing agent, little is known about its genotoxic effects, and a systematic study of the types of chromosomal alterations induced by this compound has not been performed. In this study, a combination of immunochemical, molecular and conventional cytogenetic techniques has been used to study the chromosomal alterations induced by bimolane in cultured human lymphocytes. Immunochemical staining with the CREST antibody indicated that bimolane induces micronuclei (MN) originating primarily from chromosome breakage. Interestingly fluorescence in situ hybridization (FISH) with differentially labeled chromosomes 1 and 9 centromeric probes indicated that bimolane also caused non-disjunction and polyploidy. Consistent with this, an expedited analysis of Giemsa-stained metaphase chromosomes in bimolane-treated lymphocytes revealed a high frequency of polyploidy/hyperdiploidy as well as dicentric chromosomes, and premature centromeric division (PCD). In addition, bimolane was also found to produce binucleated cells, possibly through an interference with normal functioning of intermediate filaments. As a follow-up to these studies, three different types of commercially available bimolane formulations obtained from different Chinese manufacturers were also evaluated. The effects seen with the formulated bimolane were similar to those seen with the synthesized compound. Our studies indicate that bimolane effectively induces a variety of cellular and chromosomal changes in cultured lymphocytes and that similar alterations occurring in bone marrow stem cells could contribute to the development of the secondary cancers seen in bimolane-treated patients.     

Clinical implication of p53 mutation in lung cancer

Lung cancer development involves multiple genetic abnormalities leading to malignant transformation of the bronchial epithelial cells, followed by invasion and metastasis. One of the most common changes is mutation of the p53 tumor suppressor gene. The frequency of p53 alterations in lung cancer is highest in small cell and squamous cell carcinomas. A genetic “signature” of the type of p53 mutations has been associated with carcinogens in cigarette smoke. The majority of clinical studies suggest that lung cancers with p53 alterations carry a worse prognosis, and may be relatively more resistant to chemotherapy and radiation. An understanding of the role of p53 in human lung cancer may lead to more rational targeted approaches for treating this disease. P53 gene replacement is currently under clinical investigation but clearly more effective means of gene deliver to the tumor cells are required. Novel approaches to lung cancer therapy are needed to improve the observed poor patient survival despite current therapies.     

Distribution of Microsomal Epoxide Hydrolase in Humans: An Immunohistochemical Study in Normal Tissues, and Benign and Malignant Tumours

Microsomal epoxide hydrolase is a biotransformation enzyme which is involved in the hydrolysis of various epoxides and epoxide intermediates. In the present study, its distribution was investigated in both normal human tissues and human tumours of different histogenetic origin using immunohistochemical techniques. In normal tissue, epithelial cells were more often and more intensely immunostained than mesenchymal cells. The main epithelial cell types expressing microsomal epoxide hydrolase were hepatocytes, acinus cells of the pancreas, and cells of salivary and adrenal glands. Immunostained cells of mesenchymal origin included monocytes, fibrocytes, fibroblasts, vessel endothelium, muscle cells, and cells of the reproductive system. Three patterns of expression were observed in tumour tissues: (1) moderate or strong in hepatocellular carcinomas, tumours of the adrenal gland, and theca-fibromas of the ovary; (2) inhomogeneous staining pattern of variable intensity in breast cancer, lung cancer, colorectal carcinomas, carcinoid tumours, and some tumours of mesenchymal origin; and (3) no expression in malignant melanomas, malignant lymphomas, and renal carcinomas. These data indicate that microsomal epoxide hydrolase expression is not restricted to tissue of any particular histogenetic origin. Nonetheless, immunohistochemical identification of microsomal epoxide hydrolase may be helpful in some well-defined histological settings, for example, confirmation of hepatocellular carcinoma.     

Histochemical demonstration of different types of poly-N-acetyllactosamine structures in human thyroid neoplasms using lectins and endo-β-galactosidase digestion

Summary Blood-group-related antigens expressed in papillary carcinomas and other types of neoplasm of the human thyroid glands have been shown to be carried by poly-N-acetyllactosamines containing a linear domain susceptible to endo-β-galactosidase digestion. To make clear more precisely the backbone poly-N-acetyllactosamine structures, labelled lectins specific to different types of these structures and specific to core structures with β1-6GlcNAc branching of N- and O-linked glycoproteins were employed in conjunction with prior endo-β-galactosidase digestion on formalin-fixed, paraffin-embedded neoplasms of the human thyroid glands. In papillary carcinomas,Datura stramonium agglutinin (DSA) and succinyl wheat germ agglutinin (Suc-WGA) reacted most consistently and frequently with papillary carcinomas from all the individuals examined. Pokeweed mitogen (PWM) likewise stained the cells of papillary carcinomas from all the individuals examined, but in some individuals the number of lectin-reactive cells were very small.Lycoperscion esculentum aggultinin (LEA),Solanum tuberosum agglutinin (STA),Phaseolus vulgaris agglutinin L (PHA-L) andArtocarpus integrifolia agglutinin (jacalin) similarly bound to the cancer cells from most of the individuals, and in these cases the number of reactive cells was usually much more restricted than was the case with DSA or PWM. In adenoma and other types of carcinoma, such as follicular carcinomas, these lectins specific to poly-N-acetyllactosamine exhibited slight or no reactivity with the cells, whereas PHA-L and jacalin similarly bound to the cells of adenomas and carcinomas from most of the individuals examined. Prior digestion with endo-β-galactosidase completely eliminated or markedly reduced the reactivity with PWM and LEA in papillary carcinomas. Reactivity with DSA, Suc-WGA, STA, PHA-L and jacalin was slightly reduced or not at all affected by enzyme digestion. These results confirmed that poly-N-acetyllactosamine species found in papillary carcinomas are quite different from those in other types of thyroid neoplasm, suggesting that at least three different types of poly-N-acetyllactosamine, that is, linear unbranched short and long sequences and highly branched ones are produced in these cells.     

Role of induced genetic instability in the mutagenic effects of chemicals and radiation

Recent studies have demonstrated that cells exposed to ionizing radiation or alkylating agents can develop prolonged genetic instability. Induced genetic instability is manisested in multiple ways, including delayed reproductive death, an increased rate of point mutations, and an increased rate of chromosome rearrangements. In many respects these changes are similar to the genetic instability associated with cancer and some human genetic diseases. Therefore, as with cancer cells, multiple mechanisms may be involved, some occuring in the early stages and some in the later stages. The high percentage of cells that develop induced genetic instability after exposure to stress, and the prolonged period over which the instability occurs, indicates that the instability is not in response to residual damage in the DNA or mutations in specific genes. Instead, changes affecting most of the exposed cells, such as epigenetic alterations in gene expression or chain reactions of chromosome rearrangements, are a more likely explanation. Learning more about the mechanisms involved in this process is essential for understanding the consequences of exposure of cells to ionizing radiation or alkylating agents.     

Molecular aspects of stromal-parenchymal interactions in malignant neoplasms

Carcinomas are composed of parenchymal and stromal elements, and the malignant behavior is principally dictated by the cancer cells. However, the malignant tumors not merely grow into a preexisting interstitial tissue, but they actively form a new stroma and modify their composition. Thus, the tumor stroma is significantly different from that of the neighboring tissues. Cancer cells may alter their stroma by cell-to-cell contact, soluble factors or by modification of the extracellular matrix (ECM), they induce myofibroblast differentiation and govern the desmoplastic stroma reaction. On the other hand, the stromal cells (especially the myofibroblasts) are able to modify the phenotype, invasiveness, metastatic capacity of carcinomas, typically promoting the progression. Regarding pancreatic cancer, the pancreatic stellate cells (PSCs) seem to be the key elements in the cross-talk between the parenchymal cells and the desmoplastic stroma. The tumor stroma is also rich in tumor-associated macrophages (TAM), but their role in the malignant process is contradictory and may be different in various tumor types, but most studies suggest a negative impact on the tumor growth. The relationship between the parenchymal and stromal elements is highly complex, they mutually alter their characteristics. Because the neostroma of the carcinomas largely seems to promote the invasiveness of the malignant tumors, novel therapeutic strategies are being evaluated targeting the stromal elements, with some encouraging, but still fragmentary results.     

The EGFR-GEP100-Arf6 pathway in breast cancer

Arf6 and its effector AMAP1 are overexpressed in malignant breast cancer cells, and are involved in their invasion and metastasis. We recently revealed that GEP100, a guanine nucleotide exchanging factor, is responsible for the activation of Arf6 which induces invasion and metastasis. GEP100 associated directly with ligand-activated epidermal growth factor receptor (EGFR) to be activated. Disruption of E-cadherin-mediated cell-cell adhesion is one of the major steps involved in acquisition of invasive phenotypes of most carcinomas. The EGFR-GEP100-Arf6 pathway not only activated matrix invasion activity but also perturbed E-cadherin function. GEP100 was found to be expressed in more than 80% of invasive ductal carcinomas. However, 60% of ductal carcinomas in situ were also positive for GEP100, in which GEP100 was preferentially coexpressed with EGFR in their malignant cases. Microenvionments have been highly implicated in the development of tumor malignancy. Our results reveal an aspect of the precise molecular mechanism of cancer invasion and metastasis, in which full invasiveness is not acquired just by alterations of cancer cells themselves, but their microenvironments may also play pivotal roles.     

The fate of chrysotile-induced multipolar mitosis and aneuploid population in cultured lung cancer cells

Chrysotile is one of the six types of asbestos, and it is the only one that can still be commercialized in many countries. Exposure to other types of asbestos has been associated with serious diseases, such as lung carcinomas and pleural mesotheliomas. The association of chrysotile exposure with disease is controversial. However, in vitro studies show the mutagenic potential of chrysotile, which can induce DNA and cell damage. The present work aimed to analyze alterations in lung small cell carcinoma cultures after 48 h of chrysotile exposure, followed by 2, 4 and 8 days of recovery in fiber-free culture medium. Some alterations, such as aneuploid cell formation, increased number of cells in G2/M phase and cells in multipolar mitosis were observed even after 8 days of recovery. The presence of chrysotile fibers in the cell cultures was detected and cell morphology was observed by laser scanning confocal microscopy. After 4 and 8 days of recovery, only a few chrysotile fragments were present in some cells, and the cellular morphology was similar to that of control cells. Cells transfected with the GFP-tagged α-tubulin plasmid were treated with chrysotile for 24 or 48 h and cells in multipolar mitosis were observed by time-lapse microscopy. Fates of these cells were established: retention in metaphase, cell death, progression through M phase generating more than two daughter cells or cell fusion during telophase or cytokinesis. Some of them were related to the formation of aneuploid cells and cells with abnormal number of centrosomes.     

Tumor heterogeneity: morphological, molecular and clinical implications

Malignant tumors are characterized by their great heterogeneity and variability. There are hundreds of different types of malignant tumors that harbour many oncogenic alterations. The tumor heterogeneity has important morphological, molecular and clinical implications. Except for some hematopoietic and lymphoproliferative processes and small cell infant tumors, there are not specific molecular alterations for most human tumors. In this review we summarize the most important aspects of carcinogenesis and chemoradiosensitivity of malignant cells. In this regard, some oncogenes such as neu, ras and bcl-2 have been associated with cellular resistance to treatment with anticancer agents. The knowledge of oncogenic alterations involved in each tumor can be important to correlate the morphological features, the genetic background, the prognosis and the clinical response to treatment with anticancer agents. Based on the molecular background of the tumor there are new cancer gene therapy protocols. For example using adenovirus Ela in tumors with overexpression of neu oncogene, inhibitors of tyrosine kinase specific for the PDGF receptor in glioma, inhibitors of farnesil transferase to prevent ras activity in tumors with mutations in the ras gene.     

Role of the insulin-like growth factor system in adrenocortical growth control and carcinogenesis.

Clinically silent adrenocortical adenomas are the most frequent abnormalities in the adrenal gland. In contrast, adrenocortical carcinoma is a rare tumor with an extremely poor prognosis. The factors responsible for the frequent occurrence of benign adrenocortical tumors on one hand and the rare malignant transformation on the other are not known. Several genetic alterations such as loss of imprinting or loss of heterozygosity of the 11p15 gene locus causing a strong IGF-II overexpression have been demonstrated in the majority of adrenocortical carcinomas. In addition to IGF-II overexpression, increased levels of the IGF-I-receptor and IGFBP-2 have been found in advanced human adrenocortical carcinomas, suggesting an important role for the IGF-system in adrenocortical carcinogenesis. IGFs are potent mitogens regulating growth and apoptosis through interaction with the IGF-I-receptor, and overexpression of the human IGF-I-receptor promotes ligand-dependent neoplastic transformation in a variety of different cell systems. It is evident, therefore, that high levels of IGF-II in combination with overexpression of the IGF-I-receptor can provide a significant growth advantage for adrenocortical carcinoma cells and thus contribute to the highly malignant phenotype of this rare type of cancer. Additionally, it has been shown that overexpression of IGFBP-2 can promote malignant transformation of Y1 mouse adrenocortical tumor cells through unknown IGF-independent mechanisms. As one possible mechanism, we have recently found altered expression of catalase in IGFBP-2-overexpressing tumor cells, thus implicating IGFBP-2 in influencing intracellular peroxide levels. However, since transgenic mice with IGF-II or IGFBP-2 overexpression in the adrenal gland do not show an increased frequency of adrenal tumors, IGF-II or IGFBP-2 may act as progression factors but not as initiation factors in adrenocortical tumorigenesis.     

Transfection of the EJ rasHa gene into keratinocytes derived from carcinogen-induced mouse papillomas causes malignant progression.

The development of malignant tumors in carcinogen-treated mouse skin appears to involve several genetic changes. Genetic changes which initiate the process are believed to induce alterations in the normal pattern of epidermal differentiation, resulting in the formation of benign tumors, i.e., epidermal papillomas. Subsequent changes appear to be required for the malignant conversion of papillomas to epidermal, squamous-cell carcinomas. Activation of the rasHa gene occurs frequently in chemically induced benign skin papillomas as well as squamous cell carcinomas and thus may represent one mechanism to achieve the initiation step. In the present study, we analyzed several cell lines derived from chemically induced mouse skin papillomas for the presence of transforming oncogenes by transfection of their DNA into NIH 3T3 cells. These papilloma cell lines exhibit an altered differentiation program, i.e., the ability to proliferate under culture conditions favoring terminal differentiation. When DNA from six separate cell lines was tested in the NIH 3T3 transfection assay, active transforming activity was not detected. However, when the EJ rasHa gene was introduced into three of the papilloma cell lines by DNA transfection, transfectants showed an enhanced capacity to proliferate under differentiating culture conditions and formed rapidly growing, anaplastic carcinomas in nude mice. Our findings suggest that in some papilloma cells, a genetic change distinct from rasHa activation may produce an altered differentiation program associated with the initiation step, and this genetic alteration may act in a cooperating fashion with an activated ras gene to result in malignant progression.     

Can chromatin texture predict structural karyotypic changes in diploid cells from thyroid cold nodules?

To assess the effect of a single chromosomal translocation on the nuclear phenotype of human cells, seven diploid adenomas and five diploid carcinomas of the thyroid gland were studied using quantitative nuclear morphometry. Four adenomas and three carcinomas were cytogenetically normal, whereas three adenomas and two carcinomas had a unique chromosomal translocation. A densitometric parameter discriminated adenomas from carcinomas (skewness of the optical density histogram, SODH), and tumours with and without chromosomal translocation (standard deviation of the optical density, SDODH). These results demonstrate that single chromosomal structural rearrangements produce quantifiable alterations of nuclear organisation, but that other nuclear features which do not express an aneuploid DNA content or an abnormal karyotype differentially characterise benign and malignant conditions.     

Polypeptide expression in prostate hyperplasia and prostate adenocarcinoma.

Cells were collected from prostate hyperplasias (n = 6) and prostate carcinomas (n = 6) and subjected to two-dimensional gel electrophoresis (2-DE). The resulting polypeptide patterns were analysed with the PDQUEST computer software. Malignant tumors showed significant increases in the level of expression of proliferating cell nuclear antigen (PCNA), calreticulin, HSP 90 and pHSP 60, oncoprotein 18(v), elongation factor 2, glutathione-S-transferase pi (GST-pi), superoxide dismutase and triose phosphate isomerase. In addition, decreases in the levels of tropomyosin-1 and 2 and cytokeratin 18 were observed in prostate carcinomas compared to prostate hyperplasias. This pattern of alterations is similar to that observed in other carcinomas in our previous studies. All malignant tumors showed simultaneous alterations in 5 or more of 9 markers studied, whereas only one case of benign hyperplasia showed alterations in 5 markers. The EST-data base for prostate tumors available from NCI (CGAP) was searched for the expression of the mRNAs corresponding to proteins identified in our gels. Large differences in the relative expression of mRNAs and proteins were observed. Our data show alterations in the pattem of polypeptide expression in prostate carcinomas which are similar to those observed in other carcinomas.     

Cytogenetic markers in hematoproliferative disorders

In the last decade the improvement of methods of chromosome analysis has allowed new insights into the correlation of specific chromosome changes and certain types of malignant hematologic disorders. Even if a clear-cut correlation between a certain chromosomal marker and a certain malignancy is the exception, it is well established that specific chromosome aberrations occur nonrandomly in specific tumors. Moreover, it has been shown that so-called cellular oncogenes are located on those chromosome regions which are involved in translocations and other structural chromosome abnormalities in particular malignant tumors. The significance of chromosome alterations in leukemias and lymphomas is illustrated by examples concerning well-established data, on the one hand, and findings which have still to be confirmed, on the other. This may demonstrate that human tumor cytogenetics are a dynamically and vigorously developing branch of cancer research.     

Overexpression of choline kinase is a frequent feature in human tumor-derived cell lines and in lung,prostate, and colorectal human cancers

Carcinogenesis is a long process that results in the accumulation of genetic alterations primarily in genes involved in the regulation of signalling pathways relevant for the regulation of cell growth and the cell cycle. Alteration of additional genes regulating cell adhesion and migration, angiogenesis, apoptosis, and drug resistance confers to the cancer cells a more malignant phenotype. Genes that participate in the regulation of some critical metabolic pathways are also altered during this process. Choline kinase (ChoK) has been reported to belong to the latter family of cancer-related genes. Recently, we have reported that increased activity of ChoK is observed in human breast carcinomas. Here, we provide further evidence that ChoK dysregulation is a frequent event found in a variety of human tumors such as lung, colorectal, and prostate tumors. Furthermore, a large panel of human tumor-derived cell lines also show increased ChoK activity when compared to appropriate non-tumorigenic or primary cells. These findings strongly support the role of ChoK alterations in the carcinogenic process in human tumors, suggesting that ChoK could be used as a tumor marker.