In vitro transformation induces abnormal protein thiol levels in rat liver cells

Cells from an established line of normal rat hepatocytes (IAR 20) were irradiated with 3 Gy gamma-radiation from a cobalt source to generate transformed clones. Cells from four transformed colonies were compared with the parent cell line by flow cytometry following staining with ortho-phthalaldehyde and propidium iodide to quantitate protein thiols and DNA respectively. Transformed cells exhibited an increased variability in cellular protein thiols which was most evident in G1 and S phase. The altered pattern of macromolecular thiol distribution implies early changes in redox state and/or modification of the amounts or types of sulphur-containing proteins in transformed cells.     

Early overexpression of cyclin D1 protein in mouse skin carcinogenesis

Abnormal expression of cell-cycle regulatory proteins, particularlycyclin D1, has been described in human cancers. However, thereare few reports of this kind in experimental carcinogenesismodels, which provide a framework to analyze the importanceof those alterations in early cancer development. Previous studiesfrom our laboratory showed that cyclin D1 mRNA was overexpressedin skin tumors generated in SENCAR mice by a two-stage carcinogenesisprotocol. In the study presented here, immunoprecipitation offresh tumor samples confirmed the overexpression of cyclin D1protein. We also developed an immunohistochemical techniqueto determine which cells in the lesions overexpressed the cyclinand the timing of deregulation during cancer development Surprisingly,we found that all premalignant lesions, including small incipientpapillomas, overexpressed cyclin D1, whereas normal and hyperproliferativeskin were negative. Nuclear immunostaining was detected onlyin the proliferative compartments of the tumors and showed anapparent cell-cycle-related variation. These results provideevidence for a role of cyclin D1 overexpression in mouse skincarcinogenesis and support the use of this model as an alternativeto in vitro studies to help understand the involvement of cyclinderegulation in cancer development.     

Quantitative assay for carcinogen altered differentiation in mouse epidermal cells

Basal epidermal cells can be selectively maintained as a monolayer in culture medium containing a low ionic calcium concentration of 0.01-0.10 mM. Cessation of proliferation, maturation and shedding of squamous sheets can be induced in this population by increasing the calcium concentration above 0.1 mM. Since alterations in the regulation of proliferation and differentiation are associated with epidermal carcinogenesis in vivo, it appeared reasonable that changes in the phenotypic response to calcium might follow exposure to carcinogens in vitro. Support for this hypothesis was provided by the observation that malignant epidermal cells continued to proliferate when switched from low to high calcium medium, and could thus be selected from a mixture of such cells and a large excess of normal cells which did not survive after induced differentiation. Normal primary epidermal cells were plated in low calcium medium, treated on day 3 with a chemical carcinogen, maintained for 3-9 weeks in low calcium (0.02 mM) and then switched to high calcium medium (1.4 mM). After an additional 4 weeks, surviving epithelial colonies were fixed, stained with rhodamine and counted. Treatment of cultures with 7,12-dimethylbenz[a]anthracene or N-methyl-N'-nitro-N-nitrosoguanidine yielded 4-10 fold more colonies than solvent controls. Colony number was proportional to carcinogen dose for both agents, and increased with time in low calcium prior to selection by calcium increase. Cells obtained from colonies in treated cultures demonstrated characteristic epidermal morphology and keratinization, and could be subcultured, but did not grow in agar or produce tumors in syngeneic hosts. This model system represents a quantitative assay for carcinogen altered epithelial cell differentiation and may select for an early property of preneoplastic epidermal cells.     

Chemical transformation of human embryonic nasopharyngeal epithelial cells in vitro.

The association of Epstein-Barr virus (EBV) with poorly differentiated carcinoma of the nasopharynx is well known; however, certain environmental factors, such as nitrosamines, are also important for the development of this cancer (Ho, 1975). N,N'-Dinitrosopiperazine (DNPZ) can induce nasopharyngeal carcinoma in rats and increased sister chromatid exchange frequency in human embryonic nasopharyngeal epithelial (HENE) cells. We have now demonstrated the transformation by DNPZ of HENE cells, which had a prolonged life span, anchorage-independent growth, chromosomal aberrations, tumorigenicity and morphological and ultrastructural alterations. These transformed cells might derive from the columnar epithelium of the nasopharynx, as indicated by the positive histochemical reaction with CAM 5.2 antikeratin antibody. Negative results in an immunofluorescence test for EBV nuclear antigen and Southern hybridization for EBV DNA rule out the participation of this virus in the neoplastic transformation of HENE cells by DNPZ.     

Spontaneous transformation of cultured mouse bone marrow-derived stromal cells

Bone marrow-derived stromal cells have engendered interest because of their therapeutic potential for promoting tissue vascularization and repair. When mononuclear cells isolated from mouse bone marrow were cultured in DMEM supplemented with 10% fetal bovine serum, cell populations arose that showed rapid proliferation and loss of contact inhibition. These cells formed invasive soft tissue sarcomas after i.m. injection into nude or scid mice. I.v. injection resulted in the formation of tumor foci in the lungs. The tumors were transplantable into syngeneic immunocompetent mice. Direct injection of cultured cells into immunocompetent mice also resulted in tumor formation. Karyotype analysis showed that increased chromosome number and multiple Robertsonian translocations occurred at passage 3 coincident with the loss of contact inhibition. The remarkably rapid malignant transformation of cultured mouse bone marrow cells may have important implications for ongoing clinical trials of cell therapy and for models of oncogenesis.     

Centrosome overduplication, increased ploidy and transformation in cells expressing endoplasmic reticulum-associated cyclin A2

Cyclin A2 is predominantly, but not exclusively, localized in the nucleus from G1/S transition onwards. It is degraded when cells enter mitosis after nuclear envelope breakdown. We previously showed that a fusion protein (S2A) between the hepatitis B virus (HBV) surface antigen protein and a non-degradable fragment of human cyclin A2 (Delta152) resides in the endoplasmic reticulum membranes, escapes degradation and transforms normal rat fibroblasts. The present study investigates whether cytoplasmic cyclin A2 may play a role in oncogenesis. We show that the sequestration of non-degradable cyclin A2-Delta152 by a cellular ER targeting domain (PRL-A2) leads to cell transformation when coexpressed with activated Ha-ras. REF52 cells constitutively expressing PRL-A2 are found to have a high incidence of multinucleate giant cells, polyploidy and abnormal centrosome numbers, giving rise to the nucleation of multipolar spindles. Injection of these cells into athymic nude mice causes tumors, even in the absence of a cooperating Ha-ras oncogene. These results demonstrate that, independently of any viral context, an intracellular redistribution of non-degradable cyclin A2 is capable of deregulating the normal cell cycle to the point where it promotes aneuploidy and cancer.     

Mammary-carcinoma cells in mouse liver: infiltration of liver tissue and interaction with Kupffer cells

Interactions between TA3 mammary-carcinoma cells and liver cells were studied with the electron microscope in mouse livers that had been perfused with a defined medium containing the tumour cells. Infiltration of liver tissue by the TA3 cells proceeded in the following steps. First, numerous small protrusions were extended through endothelial cells and into hepatocytes. Next, some cells had larger processes deeply indenting hepatocytes. Finally a few tumour cells became located outside the blood vessels. Two variant cell lines, TA3/Ha and TA3/St, differing in cell coat and surface charge, did not differ in the extent of infiltration. TA3/Ha cells were often encircled by thin processes of liver macrophages (Kupffer cells). Encircled cells were initially intact, but later some of them degenerated. These observations suggest that TA3/Ha cells were phagocytized by the Kupffer cells. Encirclement appeared to be inhibited after only 30 min, when many cells were still partly surrounded. Encirclement of TA3/St was much less frequent. After injection of tumour cells intra-portally in vivo, similar results were obtained, which demonstrated the validity of the perfused liver model. TA3/Ha cells formed much fewer tumour nodules in the liver than TA3/St cells.     

Malignant transformation of mouse embryo cells by estrogenic hormones in vitro

Mouse embryo cells (MEC) taken from a LACA pregnant female mouse, were cultured in RPMI 1640 medium with 20% horse serum. In the experimental group (10 bottles), the first 3 generations of MEC were treated with estradiol valerate (E) and hydroxyprogesterone caproate (P) at doses of 5.2 micrograms/ml and 261 micrograms/ml medium in the original and the 2nd generations, and at double doses in the 1st generation. In the control group (10 bottles), the MEC were cultured without E and P. The MEC in both groups was subdivided into 3 bottles from 1 every seven days. In the experimental group (44 bottles), the morphological transformation emerged in 2 bottles of MEC (the 5th and 7th generations) after 79 day's treatment by E and P. Meanwhile, the nonmorphological transformed cells (the 3rd, 5th and 7th generations) had the ability to grow in ouabain-contained medium (Anti-Oua+). It implies that the mutation had already occurred before transformation. The transformed cells had the ability to form colonies in soft-agar medium, could be agglutinated by concanavalin A and form fibrosarcoma in the subcutaneous region after inoculation. Basing on the above results, we suggest that E and P directly act as a carcinogen on the MEC and cause the latter to undergo malignant transformation.     

Telomere-driven tetraploidization occurs in human cells undergoing crisis and promotes transformation of mouse cells

Human cancers with a subtetraploid karyotype are thought to originate from tetraploid precursors, but the cause of tetraploidization is unknown. We previously documented endoreduplication in mouse cells with persistent telomere dysfunction or genome-wide DNA damage. We now report that endoreduplication and mitotic failure occur during telomere crisis in human fibroblasts and mammary epithelial cells and document the role of p53 and Rb in repressing tetraploidization. Using an inducible system to generate transient telomere damage, we show that telomere-driven tetraploidization enhances the tumorigenic transformation of mouse cells. Similar to human solid cancers, the resulting tumors evolved subtetraploid karyotypes. These data establish that telomere-driven tetraploidization is induced by critically short telomeres and has the potential to promote tumorigenesis in early cancerous lesions.     

Expression of liver phenotypes in cultured mouse hepatoma cells

Mouse hepatoma cells were established in vitro as a permanently growing line designated Hepa. The mass population and a subclone were characterized for their karyotype and their retention of liver-specific properties. An examination of 17 hepatic traits revealed that the cell lines secreted several serum proteins. The activities of a number of liver-specific enzymes, however, appeared to be absent in these cells. The identification of differentiated properties of cultured hepatoma cells permits the use of these lines in a variety of studies such as cell hybridization, biochemical analysis of tissue-specific gene products, and the modulation of expression of genes governing differentiated phenotypes. This report presents the analysis of a broad spectrum of characteristics and thereby describes one of the most fully defined hepatoma cell lines of murine origin in the literature.     

Properties of carcinogen altered mouse epidermal cells resistant to calcium-induced terminal differentiation

Eight cell lines exhibiting resistance to Ca²⁺ induced terminaldifferentiation were derived from primary mouse epidermal culturesand their properties analyzed. The lines developed either spontaneously(2 lines) or after exposure of primary cultures to carcinogensor carcinogens and tumor promoter. All but one of the lineswere of epithelial or epitheloid morphology but 3 of the 8 lineslacked desmosomes, keratin filaments and immunoprecipitablekeratin proteins, and thus could not be defined as keratinocytes.Two of the 5 keratino-cyte lines were tumorigenic in syngeneicBalb/c newborns after 4 months in medium containing 1.2 mM Ca²⁺, and 3 lines remained non-tumorigenic even after 11 monthsin 1.2 mM Ca²⁺. All three of the non-keratinizing lines weretumorigenic. Tumorigenic potential of the 5 keratinocyte linesdid not correlate with ploidy (as determined by DNA content),transglutaminase activity or growth in soft agar. However, the2 tumorigenic keratinocyte lines contained cells which stainedintensely red for gamma glutamyl transpeptidase activity, whilethe non-tumorigenic keratinocyte lines did not. Only those lineslacking desmosomes and keratin filaments grew in soft agar,but these lines were negative for gamma glutamyl transpeptidaseactivity. Ploidy and transglutaminase activity did not correlatewith tumorigenicity in these non-keratinizing lines. These resultsshow that cell lines derived from cultured mouse epidermal cellsand selected on the basis of their resistance to Ca²⁺ inducedterminal differentiation may be preneoplastic. Furthermore theassociation of additional markers with malignant change in thesecell lines depended on whether or not the cells were keratinizing.     

Tg737 inhibition results in malignant transformation in fetal liver stem/progenitor cells by promoting cell-cycle progression and differentiation arrest

Cancer stem/progenitor cells (CSPCs) may originate from the malignant transformation of normal stem cells. However, the mechanism by which normal stem cells undergo such transformation is not understood. Our previous studies provided evidence that Tg737 may play an important role in carcinogenesis of liver stem cells. In this study, we investigated the role of Tg737 in the malignant transformation of fetal liver stem/progenitor cells (FLSPCs). We inhibited Tg737 in FLSPCs using short hairpin RNA (shRNA). The microscopic observations of freshly purified Tg737 normal FLSPCs (nFLSPCs) and Tg737‐silent FLSPCs (sFLSPCs), which showed high expression levels of stem cell markers, revealed no significant morphological changes in sFLSPCs. Following RNAi of Tg737, the mRNA and protein levels of sFLSPCs decreased by 81.81% and 80.10% as shown by PCR, Western blot and immunocytochemistry analyses. Excluding apoptosis‐related effects, we found that silencing of Tg737 resulted in enhanced cell proliferation through promoting cell‐cycle progression via upregulation of cyclin D1 and cyclin B expression (P < 0.05). Silencing of Tg737 also resulted in significant arrest of cell differentiation (P < 0.05), stable expression of both albumin (ALB) and alpha fetoprotein (AFP) (P > 0.05) and quiescent ultrastructure. Assessment of cell malignant traits by transwell migration assays and by growth of xenograft tumors in athymic mice showed that reduced expression of Tg737 greatly promoted cell invasion and hepatocarcinogenesis of FLSPCs (P < 0.05). This work shows that inactivation of Tg737 may play an important role in malignant transformation of FLSPCs. © 2011 Wiley Periodicals, Inc.     

Expression and subcellular localization of cyclin D1 protein in epithelial ovarian tumour cells

The expression of cyclin D1 protein in tumour sections from 81 patients with epithelial ovarian cancer was analysed using immunohistochemistry. The tumours that overexpressed cyclin D1 in more than 10% of neoplastic cells were considered positive. Thus overexpression of cyclin D1 was observed in 72/81 (89%) of the cases examined. Protein was detected in both the nucleus and the cytoplasm in 24/81 (30%) and localized exclusively in the cytoplasm in 48/81 (59%) of the tumours. Cyclin D1 was overexpressed in both borderline and invasive tumours. There was no association between protein overexpression and tumour stage and differentiation. Furthermore, no correlation between cyclin D1 expression and clinical outcome was observed. However, in tumours overexpressing cyclin D1 (n = 72), the proportion displaying exclusively cytoplasmic localization of protein was higher in those with serous compared with non-serous histology (P = 0.004, odds ratio 4.8, 95% confidence interval 1.4-19.1). Western analysis using a monoclonal antibody to cyclin D1 identified a 36 kDa protein in homogenates from seven tumours displaying cytoplasmic only and one tumour demonstrating both nuclear and cytoplasmic immunostaining. Using restriction fragment length polymorphism polymerase chain reaction and PCR-multiplex analysis, amplification of the cyclin D1 gene (CCND1 was detected in 1/29 of the tumours demonstrating overexpression of cyclin D1 protein. We conclude that deregulation of CCND1 expression leading to both cytoplasmic and nuclear protein localization is a frequent event in ovarian cancer and occurs mainly in the absence of gene amplification.