Detection of theH-RAS oncogene in human thyroid anaplastic carcinomas

Summary We have transfected high-molecular-weight DNA from human thyroid carcinomas into murine 3T3 cells. As a result we identified several foci of morphologically distinct transformed cells in each of the tumour DNA transfected cultures. After a total of three rounds of transfection, the transformed cells were shown to form tumours in nude mice. Southern blot analysis of DNA prepared from third-round transfectants demonstrated the presence of human Alu repetitive sequences and, after hybridization with probes for known oncogenes, indicated the presence of the humanH-RAS oncogene in 3T3 cells transfected with three out of four anaplastic carcinoma DNA samples. It appears therefore that activation ofRAS genes may be an important event in the development of the anaplastic thyroid tumours.     

Cell transformation in vitro by transfer of isolated metaphase chromosomes]

Human and murine cells can be transformed in vitro following transfer of chromosomes (transfection) isolated from tumour (HeLa) or SV40-transformed (WI98VaD) human cells. An abortive transformation of Mouse cells is observed in soft-agar medium. An instability of the transformed phenotype is exhibited by the transfected human cells, following the isolation of colonies growing in soft-agar or low-serum medium. Nevertheless, two transformed cell lines (809 ch. VaD, Cl.5P and Cl.6P) could be established in culture.     

Globin gene expression in MSV-transformed fibroblasts.

The activation of globin gene expression on viral transformation of 3T3 cells was investigated. Globin mRNA was determined using a radioactive complementary DNA probe. No difference was found between 3T3 and transformed 3T3 cells. There does not therefore appear to be a random activation of extensive regions of the cellular genome.     

Cisplatin-induced genes as potential markers for thyroid cancer

Despite the uncontested role of p53 in cycle arrest/cell death after cisplatin treatment, to date the question whether wild-type p53 confers a resistant or sensitive status on the cell is still a matter of debate. Isogenic and isophenotypic human thyroid papillary carcinoma cell line variants for p53 differently expressed cycle genes after cisplatin treatment. Seven genes (CDC6-related protein, CCNC, GAS1, TFDP2, MAPK10/JNK3, WEE1, RPA1) selected after expression on an Atlas human cell cycle array were analyzed by quantitative real-time PCR. While cisplatin treatment increased their expression in p53 wild-type cells it decreased it in cells with inactivated p53 and had no or less effect on cells with mutated p53. These results show that in a well-defined system, different alterations of p53 can lead to a different regulation of genes and hence to either resistance or sensitivity to cisplatin. Moreover for the first time, MAPK10/JNK3 was identified in human thyroid cells and tissue. Four of the genes (CDC6-related protein, CCNC, GAS1 and TFDP2) were decreased in human papillary carcinoma tissues. Relevance of these genes (especially a decrease in GAS1 in thyroid papillary carcinoma) in various malignant pathologies has already been shown. These genes may be explored as new markers in advanced thyroid cancer such as metastatic and anaplastic forms displaying p53 alterations.Received 26 July 2004; received after revision 14 September 2004; accepted 26 October 2004     

The mutator phenotype theory of carcinogenesis and the complex histopathology of tumours: support for the theory from the independent occurrence of nuclear abnormality,loss of specialisation and invasiveness among occasional neoplastic lesions

The mutator phenotype theory of carcinogenesis suggests that genetic instability is an early and essential part of tumour development. This instability provides for substantially random cell-to-cell genomic variation (genomic heterogeneity) to arise among cells of individual tumours. Genetically unstable cells then produce 'successful' clones of cells with the necessary mutations for malignant behaviour. In a previous paper (Bignold L. P., Cell. Mol. Life Sci. 2002; 59: 950-958), it was pointed out that a population of cells which is heterogeneous for behaviour-related genes may well also be heterogeneous for morphology-related genes. This would result in cellular pleomorphism among cells of individual tumours, and so explain this almost universal characteristic of solid malignancies. paragraph sign If the concept of random genomic variability applies fully to the histopathology of tumours, then most tumours should show a mixture of neoplastic features, especially nuclear atypia, loss of specialised function (such as loss of production of mucus by glandular cells) and invasiveness. However, occasional lesions might be expected to occur which show these characteristics independently. That is, lesions should exist which exhibit one or two of the three characteristics of neoplasms without the other(s). paragraph sign This paper identifies, among human tumours, lesions which show independence of these characteristics. Two of the examples discussed are a Bowenoid solar keratosis that shows severe nuclear atypia, but no apparent loss of specialisation and no invasiveness. On the other hand, anaplastic small cell carcinoma of the lung often exhibits marked loss of differentiation, very aggressive invasion and metastasis, but little nuclear pleomorphism. paragraph sign These examples are considered to provide further support for the importance of the mutator phenotype to the pathogenesis of neoplasia.     

T-antigen regulated expression reduces apoptosis of Tag-transformed human myoblasts

The generation of human myogenic cell lines could potentially provide a valuable source for cell transplantation in myopathies. The dysregulation of proliferative-differentiative signals by viral oncogenes can result in the induction of apoptosis. Whether apoptosis occurred in myogenic cells expressing large T antigen (Tag) from SV40 upon differentiation was unknown. Human muscle satellite cells were transfected with two different constructs, containing either an origin-defective SV40 genome or Tag under vimentin promoter control. When differentiation was triggered, Tag expression reduced the formation of myotubes and dead cells showing apoptotic features were present. However, the cells expressing SV40 Tag under vimentin promoter control retained their capacity to form myotubes and expressed the myofibrillar proteins as myosin heavy chain and dystrophin when Tag expression was silent. Their apoptotic rate was similar to that of untransfected cells. The observation that apoptosis can be prevented by the down-regulation of Tag suggests that the programmed cell death induced in transformed cells can be reversed, and confirms the regulatory efficiency of the human vimentin promoter.     

Species-specific differences in the mitogenic activity of heparin-binding growth factors in the sera of various mammals

Sera from different mammalian species displayed great differences in mitogenic activity, as measured by stimulation of DNA synthesis in BALB/c 3T3 cells (3T3 cells). Among the sera examined, fetal bovine serum was least active, and increasing activity was detected in calf serum, human serum, rat serum and mouse serum, in that order. Rat and mouse sera exhibited extremely high mitogenic activity with 3T3 cells, but when TIG-1 human fetal lung fibroblasts were used for the DNA assay instead, the activity levels of all of the sera were lower, and the differences between them were smaller. To determine the reasons for these differences, the heparin-binding growth factors in each serum were separated on a heparin affinity column. Five peaks of DNA-stimulating activity were obtained. Three of these were found in all sera examined, with both 3T3 cells and TIG-1 cells. Two other peaks were found only with 3T3 cells; one was peculiar to rat and mouse sera, with extremely high activity in the rat, and the other was specific to fetal serum. The dependence of the activity of these peaks on the cells used for the test was confirmed using normal rat lung fibroblasts and immortalized rat kidney cells. These findings adequately explain the species-specific differences in mitogenic activity of whole sera, and the variation in activity depending on the cells used for assay of DNA synthesis.     

Species-specific differences in the mitogenic activity of heparin-binding growth factors in the sera of various mammals.

Sera from different mammalian species displayed great differences in mitogenic activity, as measured by stimulation of DNA synthesis in BALB/c 3T3 cells (3T3 cells). Among the sera examined, fetal bovine serum was least active, and increasing activity was detected in calf serum, human serum, rat serum and mouse serum, in that order. Rat and mouse sera exhibited extremely high mitogenic activity with 3T3 cells, but when TIG-1 human fetal lung fibroblasts were used for the DNA assay instead, the activity levels of all of the sera were lower, and the differences between them were smaller. To determine the reasons for these differences, the heparin-binding growth factors in each serum were separated on a heparin affinity column. Five peaks of DNA-stimulating activity were obtained. Three of these were found in all sera examined, with both 3T3 cells and TIG-1 cells. Two other peaks were found only with 3T3 cells; one was peculiar to rat and mouse sera, with extremely high activity in the rat, and the other was specific to fetal serum. The dependence of the activity of these peaks on the cells used for the test was confirmed using normal rat lung fibroblasts and immortalized rat kidney cells. These findings adequately explain the species-specific differences in mitogenic activity of whole sera, and the variation in activity depending on the cells used for assay of DNA synthesis.     

Levels of triiodothyronine and reverse triiodothyronine in the thyroid glands of man and swine at different stages of development]

3,5,3'-triiodothyronine (T3) and 3,3',5'-triiodothyronine (rT3) were measured by radioimmunoassay in saline extracts of neonates and human adult thyroid tissues and of fetuses, Piglets and adult Swine thyroid tissues. In all these extracts, T3 content was higher than rT3 content whatever the period of development. Both triiodoamino acids represent a small percentage of the iodinated protein in thyroid tissues.     

Transformation, in vitro, of cerebral hamster cells by polyoma virus]

A cell line called HCxPy was obtained in vitro by transformation of dissociated hamster brain cell cultures by polyoma virus. The first foci of transformed cells became evident 90 to 120 days after viral infection. This cell line is now at the 46th passage. The cells appear tumorigenic for hamsters after subcutaneous and intracerebral injection. They carry the polyoma virus T and cell surface antigens. Good evidence for astrocytic differentiation can be found by morphological examination of the tumours and of the cultured cells.     

Differences in in vitro incorporation of tritiated deoxycytidine and tritiated thymidine into human lymphocytes.

Human lymphocytes stimulated in vitro by PHA or PWM incorporate 3H-CdR into DNA, but less well than 3H-TdR. More 3H-CdR is incorporated in populations enriched for T cells. No 3H-CdR is used by cells stimulated by lipopolysaccharides under circumstances in which 3H-TdR is incorporated. We conclude that human T cells and B cells differ in their ability to use 3H-CdR.     

Cyclin D3 and p53 mediate sulforaphane-induced cell cycle delay and apoptosis in non-transformed human T lymphocytes

Despite experimental evidence that sulforaphane can exert chemopreventive effects, whether these effects are specific for neoplastic cells is not known. Following our previous demonstration that sulforaphane induces cell cycle arrest and apoptosis in human T lymphoblastoid Jurkat leukemia cells and increases p53 and bax protein expression, we tested sulforaphane on non-transformed phytohemagglutinin-stimulated human lymphocytes. Here, we demonstrate that sulforaphane arrested cell cycle progression in G, phase, through a decrease in the protein expression of cyclin D3. Moreover, sulforaphane induced apoptosis (and also necrosis), mediated by an increase in the expression of p53. These findings suggest that sulforaphane is a growth modulator for T cells. Our in vitro evidence that sulforaphane is active and even cytotoxic in normal as well as transformed lymphocytes raises important questions regarding its suitability for cancer chemoprevention.     

Presence of lectin-like substances in the basement membrane of the human kidney glomerulus]

Crude extracts from the human glomerular basement membranes solubilized by pepsin or bacterial collagenase agglutinate normal or transformed human cells. Cytoagglutination is inhibited by N-acetyl-osamines. These properties are reminiscent of lectins. When agglutinated cells are incubated for an additional 20 hrs. period in minimal serum free medium but in presence of these basement membrane extracts, they attach to the glass and spread out.     

High frequency of human cytomegalovirus (HCMV)-specific CD8+-T cells detected in a healthy CMV-seropositive donor

Human cytomegalovirus (HCMV) persists after infection but is controlled by cellular immune responses, particularly by CD8⁺ T cells. If infected individuals are immunosuppressed, HCMV can be reactivated. Upon testing the blood of healthy donors with human lymphocyte antigen tetramers, we found one individual with about 50 % of his CD8⁺ T cells being specific for the immunodominant pp65 epitope NLVPMVATV. Over a period of 2 years the high level of HCMV-specific T cells was maintained, and no HCMV DNA could be detected. At one timepoint, however, HCMV-specific DNA was detected, while 65 % of CD8⁺ T cells were specific for HCMV. When virus was detectable, a lower percentage of HCMV-specific CD8⁺ T cells showed interferon γ (IFN-γ) production after peptide stimulation in vitro. These data suggest that HCMV reactivation may also occur in immunocompetent persons, accompanied by the presence of HCMV-specific CD8⁺ T cells which are not producing IFNγ, and therefore potentially anergic or in vivo exhausted. Received 6 March 2002; received after revision 15 April 2002; accepted 17 April 2002     

Hormonal control of manganese transport in the mouse thyroid.

The present study deals with a possible mechanism controlling the transport of manganese (Mn), an essential trace element, from the circulation to the thyroid. Mice were pretreated with propylthiouracil (PTU) or triiodothyronine (T3), and a measurement of the thyroid:serum concentration ratio (T/S) of radioactive manganese (54Mn) was carried out. The T/S of 54Mn was greatly enhanced by PTU, but reduced by T3. Several methods were used to demonstrate that the T/S of 54Mn depends upon the level of thyroid-stimulating hormone (TSH) in the serum. First, bovine TSH was injected into mice; an increase in the T/S resulted. Secondly, serum thyroxine and T3 levels measured by radioimmunoassay (RIA) suggested that PTU produced an increase in serum TSH and T3 a decrease. However, direct measurement of mouse TSH by RIA for rat TSH failed to produce proof of any changes in TSH level, owing to poor cross-reactivity. Taking all the information into account, it is concluded that Mn-transport into the thyroid is controlled by the thyroid state.     

Endocrine cells producing regulatory peptides

Recent data on the immunolocalization of regulatory peptides and related propeptide sequences in endocrine cells and tumors of the gastrointestinal tract, pancreas, lung, thyroid, pituitary (ACTH and opioids), adrenals and paraganglia have been revised and discussed. Gastrin, xenopsin, cholecystokinin (CCK), somatostatin, motilin, secretin, GIP (gastric inhibitory polypeptide), neurotensin, glicentin/glucagon-37 and PYY (peptide tyrosine tyrosine) are the main products of gastrointestinal endocrine cells; glucagon, CRF (corticotropin releasing factor), somatostatin, PP (pancreatic polypeptide) and GRF (growth hormone releasing factor), in addition to insulin, are produced in pancreatic islet cells; bombesin-related peptides are the main markers of pulmonary endocrine cells; calcitonin and CGRP (calcitonin gene-related peptide) occur in thyroid and extrathyroid C cells; ACTH and endorphins in anterior and intermediate lobe pituitary cells, alpha-MSH and CLIP (corticotropin-like intermediate lobe peptide) in intermediate lobe cells; met- and leu-enkephalins and related peptides in adrenal medullary and paraganglionic cells as well as in some gut (enterochromaffin) cells; NPY (neuropeptide Y) in adrenaline-type adrenal medullary cells, etc.. Both tissue-appropriate and tissue-inappropriate regulatory peptides are produced by endocrine tumours, with inappropriate peptides mostly produced by malignant tumours.     

靶向CD133的RNA干扰对人肝癌SMMC-7721细胞的生长抑制作用

目的利用RNA干扰技术下调SMMC-7721肝癌细胞中CD133的表达,观察其在肝癌细胞增殖和侵袭方面的作用.方法构建CD133特异性的siRNA真核表达载体,转染SMMC-7721肝癌细胞并筛选出稳定表达siRNA的转染克隆;应用RT-PCR和Western blot检测CD133 mRNA和蛋白的表达;流式细胞仪检...     

Differences in in vitro incorporation of tritiated deoxycytidine and tritiated thymidine into human lymphocytes

Summary Human lymphocytes stimulated in vitro by PHA or PWM incorporate³H-CdR into DNA, but less well than³H-TdR. More³H-CdR is incorporated in populations enriched for T cells. No³H-CdR is used by cells stimulated by lipopolysaccharides under circumstances in which³H-TdR is incorporated. We conclude that human T cells and B cells differ in their ability to use³H-CdR.