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PubMed2001

P53 plays a protective role against UV- and cisplatin-induced apoptosis in transcription-coupled repair proficient fibroblasts.

We previously reported that transcription-coupled repair (TCR)-deficient human fibroblasts are extremely sensitive to UV-induced apoptosis and this sensitivity correlated with the induction of the p53 tumour suppressor. However, we have also found that p53 can be protective against UV-induced apoptosis. Thus, prior to this study, it was not clear whether the induction of p53 in TCR-deficient fibroblasts contributed to their death. To address this issue, we have expressed human papillomavirus E6 (HPV-E6) in primary fibroblasts derived from patients affected with xeroderma pigmentosum (complementation groups A, B and C) and Cockayne syndrome (complementation group B). We found that TCR-deficient (XP-A, XP-B and CS-B) fibroblasts were more sensitive than TCR-proficient cells (XP-C and normal) to both UV light and cisplatin treatment and this increase in sensitivity was not p53 dependent. Importantly, HPV-E6 expression increased the sensitivity of TCR-proficient normal and XP-C fibroblasts to UV- and cisplatin-induced apoptosis. This increase in sensitivity correlated with a decrease in the capacity of HPV-E6 expressing cells to recover mRNA synthesis following UV-irradiation. Therefore, we propose that p53 protects against UV- and cisplatin-induced apoptosis in a TCR-dependent manner and that p53 does not contribute strongly to the induction of apoptosis in TCR-deficient fibroblasts.

Apoptosis↗

PubMed2007

Proinflammatory cytokine synthesis by mucosal fibroblasts from mouse colitis is enhanced by interferon-gamma-mediated up-regulation of CD40 signalling.

Gut mesenchymal fibroblasts form complex phenotypical and functional populations. They participate actively in homeostatic maintenance of the extracellular matrix, epithelial barrier function, repair mechanisms and leucocyte migration. In inflammation, they become activated, change matrix expression and synthesize proinflammatory mediators. Subpopulations of mucosal fibroblasts express CD40 and the aim of this study was to define its role in their proinflammatory function. Stable primary fibroblast lines derived from normal mouse colon and inflamed colon from CD4(+) CD45RB(high)-transplanted SCID mice were used as models to explore the role of mucosal fibroblast CD40 in the inflammatory process. Phenotype correlated with in situ fibroblast phenotype in the tissues of origin. Lines from both sources co-expressed CD40 and Thy1.2 independently of alpha-smooth muscle actin. A subpopulation of CD40(+) fibroblasts from normal colon expressed CD40 at high levels and expression was enhanced by interferon (IFN)-gamma treatment, whereas all CD40(+) fibroblasts from colitis expressed at low levels and expression was unaffected by IFN-gamma treatment. Despite lower-level expression of CD40 by cells from colitis, they secreted constitutively interleukin (IL)-6 and C-C chemokine (CCL)2. Ligation of CD40 enhanced secretion of these mediators and induced secretion of CCL3. CD40 in cells from colitis was more responsive to ligation than CD40 on cells from normal tissue and this sensitivity was amplified selectively by the action of IFN-gamma. We conclude that the inflammatory milieu in colitis induces long-lasting changes in phenotype and proinflammatory function in colonic fibroblasts. In particular, proinflammatory signalling from fibroblast CD40 is amplified synergistically by the Th1 effector T cell cytokine, IFN-gamma.

Animals↗

PubMed2002

[Osteogenic potential of fibroblasts with reconstituted telomerase activity].

OBJECTIVE: To prevent the senescence of 'seed cells' for tissue engineering, the life span of human fibroblasts is extended by reconstitution of telomerase activity, and the osteogenic potential of these fibroblasts are tested. METHODS: The pGRN145 plasmids encoding human telomerase reverse transcriptase (hTERT) were introduced into the normal human primary fibroblasts by electroporation. Telomerase activity was analyzed by TRAP-PCR assay. The beta-galactosidase stain was used to indicate the signs of cell senescence. The hTERT positive fibroblasts were then induced to form bone nodules. The bone nodules were stained by tetracycline and Alizarin Red S. RESULTS: Stable telomerase activity could be detected in the transfected fibroblasts and no signs of cell senescence were found in the fibroblasts cultured for more than 50 doublings. The hTERT positive fibroblasts could form bone nodules when they were cultured in vitro induced by bone morphogentic protein 2 and tumor necrosis factor-alpha. CONCLUSION: The fibroblasts with reconstituted telomerase activity reserve their osteogenic potential.

Cells, Cultured↗

PubMed2005

Amphiregulin expression in human mast cells and its effect on the primary human lung fibroblasts.

BACKGROUND: Amphiregulin is a member of the epidermal growth factor family and has been shown to stimulate the proliferation of human keratinocytes in an autocrine manner. OBJECTIVE: The aim of the present study was to examine the expression change of growth factors, especially amphiregulin, in human mast cells induced by IgE cross-linking. METHODS: Microarray analysis and RT-PCR were used to analyze the gene expression profile of human cord blood-derived mast cells (CBMCs) stimulated with IgE cross-linking. Protein secretion in the supernatants of CBMCs was measured by means of ELISA. Double-immunofluorescence staining was used to analyze the expression in the lung mast cells. RESULTS: Of the 64 different growth factor genes analyzed, 5 were found to be substantially upregulated. Among them, amphiregulin mRNA was induced by 44-fold in CBMCs on activation through IgE cross-linking. Secretion of amphiregulin protein was evident in CBMCs 8 hours after stimulation. Amphiregulin was also expressed in human lung mast cells from patients with asthma, as demonstrated by means of double-immunofluorescence staining. Amphiregulin promoted the proliferation of the primary human lung fibroblasts, and amphiregulin-treated primary human lung fibroblasts showed a marked increase in the expression of c-fos , a proto-oncogene that facilitates or is required for the proliferation of a wide variety of cells. CONCLUSION: Human CBMCs secreted amphiregulin on IgE cross-linking, and the amphiregulin induced proliferation of primary human lung fibroblasts. These data suggest that local release of amphiregulin by human mast cells could play an important role in lung fibrosis by promoting the proliferation of primary human lung fibroblasts.

Amphiregulin↗

PubMed1996

Microtubule-associated protein tau in human fibroblasts with the Swedish Alzheimer mutation.

The principal neuropathological hallmarks of Alzheimer's disease (AD) are plaques containing amyloid beta peptide (Abeta) and tangles with hyperphosphorylated tau. Tau is predominantly found in the nervous system but has been reported in fibroblasts from individuals with and without AD. Abeta is also found outside the nervous system and is released three times more from cultured fibroblasts carrying the Swedish Alzheimer mutation in the amyloid precursor protein (APP) gene. In the present study, we determined tau levels in fibroblasts from carriers of the Swedish Alzheimer mutation and controls. We also characterized the expression of tau in these cells. Primary fibroblast cell lines from six individuals with and six without the mutation were investigated. ELISA measurements showed no statistically significant difference in tau levels between mutation-carrying cell lines and controls. On Western blot, four bands in the range of 47-67 kDa, corresponding to traditional tau isoforms, were detected with the Tau-l and AT 120 antibodies. Furthermore, four bands between 110-125 kDa were detected. We thus conclude that increased levels of Abeta do not seem to increase the levels of tau in human fibroblasts. We also suggest that several of the traditional tau isoforms as well as isoforms of higher molecular weights, big tau, are expressed in this cell type.

Aged↗

PubMed2005

Enhanced angiotensin II-mediated effects in fibroblasts of patients with familial hypercholesterolemia.

OBJECTIVE: Familial hypercholesterolemia (FH) is characterized by a high incidence of coronary heart disease. Evidence suggests an important role for angiotensin II (AngII) in the fibrotic response to tissue injury, and in promoting myocardial hypertrophy via paracrine mechanisms mediated by fibroblasts. We sought to determine whether AngII promotes proliferative and pro-atherogenic responses in FH patients. METHODS: We used primary fibroblasts -- from five patients with heterozygous FH and five control subjects (C) -- to study AngII-induced cell growth, intracellular calcium fluxes, and expression/release of matrix components and pro-inflammatory peptides [transforming growth factor-beta1 (TGFbeta1) and endothelin-1 (ET-1)] and metalloproteinases involved in plaque remodeling and vulnerability. RESULTS: AngII stimulated cell replication (5.1 +/- 0.03 versus 3.2 +/- 0.04 cells/50 cells per well, P < 0.001), and induced a larger increase in intracellular calcium content in FH cells than in C cells, in a dose-dependent fashion (mean difference = 76 nmol/l, P < 0.001). Similarly, TGFbeta1 and ET-1 expression and release were potentiated (after 24-h incubation with 1 micromol/l AngII: TGFbeta1 was 190 +/- 12 in C and 376 +/- 9 pg/ml per 10(6) cells in FH, and ET-1 was 93 +/- 5 in C and 192 +/- 7 pmol/ml per 10(6) cells in FH; P < 0.001 for both). AngII-induced release of the metalloproteinases MMP-1 and MMP-2 was also increased in FH versus C cells (0.52 +/- 0.04 versus 0.36 +/- 0.05 and 24 +/- 4 versus 13 +/- 3 ng/mg protein with 1 micromol/l AngII). These enhanced responses were likely due to an increased angiotensin receptor 1 (AT1) expression in cells from FH patients induced by AngII, and were prevented by pretreating cells with the selective AT1 antagonist irbesartan. CONCLUSIONS: These findings show that some AngII-mediated pathways are enhanced in FH subjects irrespective of the presence of low-density lipoprotein (LDL), thus contributing to the development and progression of atherosclerosis in these patients.

Adult↗

PubMed2005

FGF-2, IL-1beta and TGF-beta regulate fibroblast expression of S100A8.

Growth factors, including fibroblast growth factor-2 (FGF-2) and transforming growth factor-beta (TGF-beta) regulate fibroblast function, differentiation and proliferation. S100A8 and S100A9 are members of the S100 family of Ca2+-binding proteins and are now accepted as markers of inflammation. They are expressed by keratinocytes and inflammatory cells in human/murine wounds and by appropriately activated macrophages, endothelial cells, epithelial cells and keratinocytes in vitro. In this study, regulation and expression of S100A8 and S100A9 were examined in fibroblasts. Endotoxin (LPS), interferon gamma (IFNgamma), tumour-necrosis factor (TNF) and TGF-beta did not induce the S100A8 gene in murine fibroblasts whereas FGF-2 induced mRNA maximally after 12 h. The FGF-2 response was strongly enhanced and prolonged by heparin. Interleukin-1beta (IL-1beta) alone, or in synergy with FGF-2/heparin strongly induced the gene in 3T3 fibroblasts. S100A9 mRNA was not induced under any condition. Induction of S100A8 in the absence of S100A9 was confirmed in primary fibroblasts. S100A8 mRNA induction by FGF-2 and IL-1beta was partially dependent on the mitogen-activated-protein-kinase pathway and dependent on new protein synthesis. FGF-2-responsive elements were distinct from the IL-1beta-responsive elements in the S100A8 gene promoter. FGF-2-/heparin-induced, but not IL-1beta-induced responses were significantly suppressed by TGF-beta, possibly mediated by decreased mRNA stability. S100A8 in activated fibroblasts was mainly intracytoplasmic. Rat dermal wounds contained numerous S100A8-positive fibroblast-like cells 2 and 4 days post injury; numbers declined by 7 days. Up-regulation of S100A8 by FGF-2/IL-1beta, down-regulation by TGF-beta, and its time-dependent expression in wound fibroblasts suggest a role in fibroblast differentiation at sites of inflammation and repair.

3T3 Cells↗

PubMed1992

Demonstration and localization of growth hormone receptor in human skin and skin fibroblasts.

Clinical evidence suggests that skin is responsive to GH status in vivo. We sought to demonstrate the presence of GH receptors in human skin and in cultured skin fibroblasts using the techniques of immunohistochemistry and northern blotting. Human foreskin was obtained at surgery for preparation of sections and primary fibroblast cultures. Skin sections and fibroblast monolayers were immunostained using a monoclonal antibody which recognizes the hGH receptor (MAb 263). Positive immunoperoxidase staining was seen in all epidermal layers except the stratum corneum, in dermal sweat and sebaceous glands, and in dermal fibroblasts. In cultured fibroblasts capping of surface GH receptor was observed after aqueous formaldehyde fixation, whereas fixation in Carnoy's solution resulted in granular cytoplasmic staining. Fibroblast poly A+ RNA was prepared from cultured skin fibroblasts, separated by denaturing agarose gel electrophoresis, blotted onto nitrocellulose, and hybridized to a 32P-labeled, 847 base pair (bp) hGH receptor complementary DNA (cDNA) clone. Human liver and non-pregnant rabbit liver total RNA were used as controls. Fibroblast poly A+ RNA contained a single hybridizing species of approximately 5.2 kilobase. Human liver total RNA also contained a single hybridizing species of 4.9 kilobase. We have demonstrated the presence of GH receptor protein in human skin and growth hormone receptor mRNA and protein in cultured human skin fibroblasts. These observations suggest that GH may indeed have a direct role in modulating keratinocyte and fibroblast function.

Adolescent↗

PubMed1990

The relationship of excess copper accumulation by fibroblasts from the brindled mouse model of Menkes disease to the primary defect.

Fibroblasts from the brindled mouse model of Menkes disease are known to accumulate excess copper. Most of the copper in the cytosol of these fibroblasts is bound to metallothionein (MT), which is elevated in Menkes or brindled mouse fibroblasts. Copper accumulation by normal fibroblasts containing excess MT was examined to determine if the excess copper accumulation phenotype was secondary to excess MT or associated with the primary defect in fibroblasts from the brindled mice. MT was induced in normal fibroblasts by copper, zinc or dexamethasone to levels comparable with those in brindled mice fibroblasts, as determined by radioimmunoassays. Normal fibroblasts containing excess MT accumulate copper normally, i.e. they do not exhibit the excess copper accumulation phenotype. Consistent with this result, copper efflux from normal fibroblasts containing excess MT was also normal. The data suggest that one function of the protein associated with the primary defect is to help determine how much copper is taken up and retained by fibroblasts and other cell types exhibiting the excess copper phenotype in Menkes disease. The capacity of this protein is apparently exceeded in normal fibroblasts if serum or albumin is not present extracellularly to limit total copper uptake. Consistent with a defect in an intracellular protein, the kinetics of copper transport by brindled mice fibroblasts were found to be normal.

Animals↗

PubMed1996

Induction of cytokines and ICAM-1 by proinflammatory cytokines in primary rheumatoid synovial fibroblasts and inhibition by N-acetyl-L-cysteine and aspirin.

The role of transcription factor NF-kappa B in the induction of cytokines and ICAM-1 upon stimulation with proinflammatory cytokines, IL-1 and tumor necrosis factor (TNF)-alpha was investigated in primary synovial fibroblasts obtained from patients with rheumatoid arthritis (RA). Nuclear translocation of NF-kappa B was demonstrated after 30 min of treatment with IL-1 or TNF-alpha. Thereafter, the production of several cytokines including granulocyte macrophage colony stimulating factor, IL-6 and IL-8, that are known to be abundantly produced in the synovial cavity of RA patients, was greatly augmented. Similarly, cell surface expression of ICAM-1 was induced by the IL-1 or TNF-alpha treatment. Since expression of these genes is induced in rheumatoid synovial tissue, this experimental system is considered to represent the in vivo situation of RA pathophysiology. Using this cell culture system we attempted to modulate the intracellular signaling cascade for NF-kappa B activation and examined the effects of N-acetyl-L-cysteine (NAC) and acetylsalicylic acid (aspirin), which were previously reported to inhibit NF-kappa B activation. Pretreatment of the primary synovial fibroblasts with NAC inhibited nuclear translocation of NF-kappa B. Subsequently, the induction of these cytokines and ICAM-1 was considerably suppressed. On the other hand, pretreatment with aspirin blocked these phenomena only partially. These observations indicate the pivotal role of NF-kappa B in RA pathogenesis thus highlighting the possibility of a novel therapeutic strategy.

Acetylcysteine↗

PubMed2002

Analysis of Werner's expression within the brain and primary neuronal culture.

Werner's syndrome is a genetic progeria disorder caused by mutation of the Werner gene (WRN). The presence of mutations in the WRN gene is believed to result in a deleterious loss of normal WRN function, which has been best characterized for its role as a DNA helicase and exonuclease. The WRN gene is known to be expressed within the central nervous system, with Werner's syndrome associated with several neuropathological abnormalities including brain atrophy, gliosis and extensive cytoskeletal abnormalities. While WRN has been intensely investigated in primary fibroblast and fibroblast cell lines, at present little is known about the normal expression pattern of the WRN protein in the brain or primary neuronal cultures. In the present study we demonstrate that WRN is expressed throughout the brain, and is present in both neurons and glia. Similarly, WRN is present in both primary neurons and glia in cell culture, with extensive immunoreactivity present in the neuritic processes or neurons. Analysis of WRN RNA revealed that WRN was expressed at its highest levels in brain tissue from embryonic tissue, undergoing a biphasic pattern expression from early post-natal period into adulthood. Taken together, these data indicate that WRN is present in the cells of the brain, expressed throughout primary neuronal cells in culture, possibly playing a developmental role in the central nervous system.

Animals↗

PubMed1995

Long-term delivery of a lysosomal enzyme by genetically modified fibroblasts in dogs.

We have evaluated the feasibility and efficacy of intraperitoneal implants (neo-organs) for protein delivery in large animals. Skin biopsies were taken from four healthy dogs. Primary fibroblast cultures were transduced with a retroviral vector coding for the human beta-glucuronidase. One to six lattices each containing 10(9) skin fibroblasts were implanted into the omentum of the donor animal. Laparotomies performed at regular intervals showed vascularized neo-organs without local inflammation. Human beta-glucuronidase levels equivalent to 0.8 to 3.1% of the endogenous canine activity were detected for up to 340 days on liver biopsy samples. These results indicate that neo-organs can be considered for the long-term delivery of therapeutic proteins or enzymes in humans.

Animals↗

PubMed2000

Stricture formation in Crohn's disease: the role of intestinal fibroblasts.

OBJECTIVE: To determine whether intestinal fibroblasts in patients with Crohn's disease (CD) have an enhanced capacity to reorganize collagen and thus cause stricture formation. SUMMARY BACKGROUND DATA: Stricture formation is a characteristic feature of CD that may distinguish it from other forms of inflammatory bowel disease. Methods Fibroblasts were obtained at surgery from the colon and ileum of patients with CD and ulcerative colitis (UC) and control patients. Primary fibroblast cultures were obtained by explant technique. Fibroblast contractile activity was measured using fibroblast-populated collagen lattices (FPCLs), in which the cultured fibroblasts were seeded in free-floating collagen gel matrices that they reorganize and contract. Fibroblast contractile activity was measured as the reduction of surface area (mm2) of collagen gel matrix at 24-hour intervals for 1 week. RESULTS: Fibroblasts from patients with CD displayed enhanced capacity to contract FPCL when compared to UC and control fibroblasts. This activity was maximal in fibroblasts recovered from strictured regions in CD. Fibroblasts from patients with UC had a contractile capacity similar to that of controls. Hydrocortisone inhibited this in vitro contractile activity in a dose-dependent manner. CONCLUSIONS: Intestinal fibroblasts in CD possess enhanced capacity for collagen reorganization and contractile activity in vitro. This activity may be responsible for stricture formation in CD.

Cell Line↗

PubMed2005

Preclinical evaluation of transcriptional targeting strategies for carcinoma of the breast in a tissue slice model system.

INTRODUCTION: In view of the limited success of available treatment modalities for metastatic breast cancer, alternative and complementary strategies need to be developed. Adenoviral vector mediated strategies for breast cancer gene therapy and virotherapy are a promising novel therapeutic platform for the treatment of breast cancer. However, the promiscuous tropism of adenoviruses (Ads) is a major concern. Employing tissue specific promoters (TSPs) to restrict transgene expression or viral replication is an effective way to increase specificity towards tumor tissues and to reduce adverse effects in non-target tissues such as the liver. In this regard, candidate breast cancer TSPs include promoters of the genes for the epithelial glycoprotein 2 (EGP-2), cyclooxygenase-2 (Cox-2), alpha-chemokine SDF-1 receptor (stromal-cell-derived factor, CXCR4), secretory leukoprotease inhibitor (SLPI) and survivin. METHODS: We employed E1-deleted Ads that express the reporter gene luciferase under the control of the promoters of interest. We evaluated this class of vectors in various established breast cancer cell lines, primary breast cancer cells and finally in the most stringent preclinical available substrate system, constituted by precision cut tissue slices of human breast cancer and liver. RESULTS: Overall, the CXCR4 promoter exhibited the highest luciferase activity in breast cancer cell lines, primary breast cancer cells and breast cancer tissue slices. Importantly, the CXCR4 promoter displayed a very low activity in human primary fibroblasts and human liver tissue slices. Interestingly, gene expression profiles correlated with the promoter activities both in breast cancer cell lines and primary breast cancer cells. CONCLUSION: These data suggest that the CXCR4 promoter has an ideal 'breast cancer-on/liver-off' profile, and could, therefore, be a powerful tool in Ad vector based gene therapy or virotherapy of the carcinoma of the breast.

Adenoviridae↗

PubMed2000

Regulation of collagenase, stromelysin, and urokinase-type plasminogen activator in primary pterygium body fibroblasts by inflammatory cytokines.

PURPOSE: To examine the expression patterns of extracellular matrix degrading enzymes in cultured primary pterygium body fibroblasts activated by cytokines and growth factors potentially derived from ocular surface epithelial cells and tears. METHODS: EGF, TGF-alpha, PDGF-BB, IL-1beta, bFGF, TGF-beta1, TNF-alpha, or IL-6 were added at 10 ng/ml to early passaged primary pterygium body fibroblasts (PBF) or normal human conjunctival fibroblasts (HJF) in a serum-free medium. Expression of transcripts and proteins of MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1, TIMP-2, and uPA was determined by Northern hybridization, ELISA, and Western blotting, respectively. Gelatin and casein zymographies were performed in their serum-free conditioned media with or without enzyme inhibitors to determine the activity of MMP-2 and -3, respectively. RESULTS: IL-1beta and TNF-alpha dramatically increased the mRNA and protein expression of MMP-1 and MMP-3 in cultured PBF when compared to normal HJF and to their nonstimulated counterparts cultured in a serum-free medium. EGF and TGF-alpha also upregulated MMP-3 in PBF when compared to HJF. The transcript levels of MMP-2 were high but stable for the two cell types regardless of the cytokine treatment. Both TIMP-1 and TIMP-2 expressions were not influenced by the cell type or the cytokine treatment. MMP-9 was not expressed in either of these two types of fibroblasts. Both IL-1beta and TNF-alpha induced a significant decrease in uPA expression in PBF, whereas bFGF induced a slight increase in both HJF and PBF. CONCLUSIONS: Chronic inflammatory stimulation by IL-1beta and TNF-alpha, which potentially can be derived from the ocular surface and tears, may be responsible for increased expression of MMPs in cultured PBF. These data have clinical implications on progression of pterygium and recurrence associated with incomplete excision of primary PBF under the influence of ocular surface inflammation. Suppression of intraoperative and postoperative inflammation may be a new strategy to prevent pterygium recurrence.

Blotting, Northern↗

PubMed1990

Mutation eliminating mitochondrial leader sequence of methylmalonyl-CoA mutase causes muto methylmalonic acidemia.

Methylmalonyl-CoA mutase (EC 5.4.99.2) is a mitochondrial matrix enzyme whose activity is deficient in the inherited disorder methylmalonic acidemia. Previous studies on primary fibroblast cell lines from patients with methylmalonic acidemia have delineated a variety of biochemical phenotypes underlying this disorder. One cell line with primary mutase apoenzyme deficiency exhibited a particularly unusual phenotype; it expressed an abnormally small and unstable immunoreactive protein, which was not imported by mitochondria. We now report cloning and sequencing of the cDNA encoding this mutant protein. The mutation is a single base change, a cytosine----thymine transition, which introduces an amber termination codon at position 17 within the mitochondrial leader sequence. The immunoreactive protein produced by these cells reflects translation from AUG codons downstream from this termination codon and, hence, lacks a mitochondrial leader peptide. This mutation represents a complex prototype for a class of mutations in which absence of the mitochondrial targeting sequence leads to absence of a functioning gene product.

Amino Acid Metabolism, Inborn Errors↗

PubMedDate not supplied

Adenosine deaminase complexing protein (ADCP): a transformation sensitive protein with potentials of a cancer marker.

Several observations by independent investigators in the past have indicated that adenosine deaminase complexing protein (ADCP), present in considerable quantities in certain human tissues, was absent or decreased in the cancers originated from them. During the present study, electrophoretic analysis of adenosine deaminase (ADA) isozymes and radioimmunoassay for ADCP in the primary fibroblasts and the transformed as well as certain tumor derived cell lines have demonstrated that ADCP present in large quantities in the primary cells was absent or nearly absent in the transformed or tumor-derived cell lines. Though the mechanisms involved are not yet clear, the above observations indicate that ADCP has the potentials of a useful marker in the studies on transformed cells and cancer tissues.

Adenosine Deaminase↗

PubMed1997

Novel strategies for eutherian x marsupial somatic cell hybrids: mapping the genome of Monodelphis domestica.

Two hundred thirty-seven independent somatic cell hybrids have been obtained between opossum (Monodelphis domestica) splenocytes, bone marrow cells, or primary fibroblasts, and HPRT-deficient or TK-deficient Chinese hamster, mouse, American mink, or common vole fibroblast lines. Because extreme segregation and fragmentation of marsupial chromosomes commonly occurs in eutherian x marsupial somatic cells hybrids, we developed a rapid primary screening method that enables the identification of primary clones containing a large amount of opossum DNA 20-25 d after fusion. This method, which depends on in situ hybridization of biotin-labeled total opossum DNA on interphase nuclei of hybrid cells fixed on the bottom of microwell plates, was used to screen the 237 hybrid clones; 52 of them had a substantial amount of opossum DNA. G-banding and in situ hybridization of biotin-labeled total opossum DNA on metaphase spreads of the clones enabled identification of 17 hybrid clones containing from two to seven intact chromosomes of M. domestica on the background of Chinese hamster or vole chromosomes. The hybrid clones with intact opossum chromosomes are used in a panel constructed for mapping the opossum genome. Initial mapping results from these clones have led to the tentative assignment of GPI and GOT1 to chromosome 1; 6PGD to chromosome 4; LDHA to chromosome 5; LDHB to chromosome 8; and PGK and G6PD to the X chromosome. On the basis of indirect evidence we also tentatively assigned HPRT to the X chromosome and TK to chromosome 5 of M. domestica. These are the first tentative chromosomal assignments by any technique for this species.

Animals↗