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Biomedical subjects

L I Huschtscha

Publications and source records attributed to L I Huschtscha.

16 recordsLinked to original sources
PubMed2001

Effects of simian virus 40 T-antigens on normal human mammary epithelial cells reveal evidence for spontaneous alterations in addition to loss of p16(INK4a) expression.

Under standard culture conditions, normal human mammary epithelial cells (HMECs) divide a limited number of times before proliferation ceases in a growth-arrested state referred to as selection. Cells that have undergone spontaneous loss of p16(INK4a) expression due to hypermethylation of the p16(INK4a) CpG island emerge from selection and proliferate for an extended, but limited, period before senescence. Here we show, as expected, that selection was bypassed by expression of SV40 large T-antigen proteins containing an intact pRb-binding domain in preselection cells. These cells were immortalized with high efficiency (seven of nine separate cultures). Also as expected, postselection cells were immortalized by expression of the human papillomavirus-16 E6 oncoprotein (four of four cultures), which inactivates p53 protein. In contrast, we found that expression of SV40 large T-antigen protein, which also inactivates p53, was poorly maintained in postselection cultures due to its growth-suppressive effects; consequently, these cells became immortalized at low efficiency (one of 11 cultures). Reexpression of p16(INK4a) in postselection HMECs by the demethylating agent, 5-azacytidine, or transfection of a p16(INK4a) expression plasmid did not restore the ability of these cells to undergo SV40-induced transformation. Postselection HMECs are a widely used in vitro model system, but these observations indicate they have undergone changes in gene expression in addition to loss of p16(INK4a) expression.

Animals↗
PubMed1999

Telomerase-negative immortalized human cells contain a novel type of promyelocytic leukemia (PML) body.

Telomerase-negative immortalized human cells maintain their telomeres by a mechanism known as alternative lengthening of telomeres (ALT). We report here that ALT cells contain a novel promyelocytic leukemia (PML) body (ALT-associated PML body, APB). APBs are large donut-shaped nuclear structures containing PML protein, telomeric DNA, and the telomere binding proteins human telomere repeat binding factors 1 and 2. Immunostaining showed that APBs also contain replication factor A, RAD51, and RAD52, proteins involved in DNA synthesis and recombination. During immortalization, APBs appeared at exactly the same time as activation of ALT. APBs were found in ALT tumors and cell lines but not in mortal cell strains or in telomerase-positive cell lines or tumors.

Cell Aggregation↗
PubMed1999

p16(INK4a) and the control of cellular proliferative life span.

Normal somatic cells have a limited proliferative capacity in vitro: after a finite number of cell divisions they eventually enter a non-proliferative state referred to as senescence. Senescence is thought to be a major tumor suppressor mechanism, and many cancers contain cells that have escaped from senescence and become immortalized. The role of telomerase activation in immortalization is currently attracting considerable attention, but immortalization is often associated with other changes including loss of normal function of the tumor suppressor locus, INK4a/ARF. Two proteins, p16(INK4a) and p14(ARF), are encoded by this locus. Here we focus on p16(INK4a) and review accumulating evidence that loss of p16(INK4a) function may be involved in escape from the normal limits on cellular proliferative life span.

Cell Division↗
PubMed1998

Loss of p16INK4 expression by methylation is associated with lifespan extension of human mammary epithelial cells.

Inactivation of p16INK4 tumor suppressor gene function is frequently observed in breast cancer. We examined p16INK4 expression in human mammary epithelial cell (HMEC) cultures established from four normal donors. Normal HMECs divide a limited number of times before proliferation ceases in a state referred to as selection (or M0). The cell subpopulation that emerges spontaneously from selection undergoes a further limited period of proliferation before senescence. By immunofluorescence and Western blot analysis of four independent cultures, we have shown loss of p16INK4 expression in postselection HMECs. In contrast, p16INK4 was present in both early and late passage fibroblasts from the same individuals. Bisulfite genomic sequencing revealed extensive methylation of the p16INK4 CpG island in post- but not preselection cells. Thus, the extended period of growth observed in postselection HMECs is associated with hypermethylation of the p16INK4 CpG island and loss of p16INK4 expression. Although postselection HMECs are widely considered to be normal, these data indicate that they have sustained an epigenetic alteration.

Breast↗
PubMed1996

Characteristics of cancer cell death after exposure to cytotoxic drugs in vitro.

The characteristics of cell death were investigated after exposure of CCRF-CEM.f2 cells to five drugs over a broad concentration range; these were the glucocorticoid dexamethasone (DXM), the mitotic inhibitor vincristine (VIN) and three antimetabolites, methotrexate (MTX), 5'-fluoro-2'-deoxyuridine (FUdR) and 5'-fluorouracil (5-FU). Drug-treated cells were monitored for cell death mechanisms at different times by examining the pattern of DNA degradation, cell morphology and flow cytometric profile, together with effects on cell growth over 72 h. At growth-inhibitory drug concentrations, the first changes were cell cycle perturbations detectable after 4-6 h of drug exposure. The appearance of features characteristic of apoptotic cell death was noted after all drug treatments in the CCRF-CEM.f2 cell line, but the pattern and kinetics varied considerably. VIN induced apoptotic changes by 12 h, while DXM treatment caused apoptosis only after 48 h. Both MTX and FUdR induced morphological changes characteristic of apoptosis at least 24 h before internucleosomal DNA cleavage, which was detectable only after 48 h. In contrast, 5-FU did not cause internucleosomal DNA cleavage by 48 h at any concentration, despite the presence of morphologically apoptotic cells 24 h earlier. These data suggest that disruption of the cell cycle caused by drug treatment may be the common trigger initiating the drug-specific apoptotic sequence of dying cells.

Antineoplastic Agents↗
PubMed1994

Identification of apoptotic and necrotic human leukemic cells by flow cytometry.

Glucocorticoids kill human leukemic cells, CCRF-CEM.f2, by apoptosis. Cell death is preceded by DNA fragmentation into nucleosomal subunits which can be evaluated by DNA gel electrophoresis or by determining the proportion of cleaved DNA in whole cell lysates. Hyperthermic treatment of CCRF-CEM.f2 cells induces necrotic cell death which is characterized by a different DNA gel electrophoretic pattern. However, these techniques cannot distinguish the individual contributions of both forms of death in a heterogeneous population. Therefore, we have developed a flow cytometric method that readily distinguishes apoptotic and necrotic cells on the basis of propidium iodide staining and cellular light scatter characteristics. This method can be used to analyze factors influencing the mechanisms of cell death after cytotoxic drug treatment.

Apoptosis↗
PubMed1993

Sodium butyrate induces apoptosis in human colonic tumour cell lines in a p53-independent pathway: implications for the possible role of dietary fibre in the prevention of large-bowel cancer.

The purpose of this study was to determine whether cultured colonic adenoma and carcinoma cells undergo apoptosis (programmed cell death) in vitro and whether specific growth and dietary factors, thought to be involved in the control of growth and differentiation of human colonic cells, could induce cell death through apoptosis. In cell lines originating from 6 colorectal adenomas and 7 carcinomas, spontaneous apoptosis was observed. Sodium butyrate, a naturally occurring fatty acid, is present in the human large bowel in millimolar amounts as a result of bacterial fermentation of dietary fibre. Sodium butyrate, at physiological concentrations, induced apoptosis in 2 adenoma cell lines, RG/C2 and AA/Cl, and in the carcinoma cell line PC/JW/FI. In contrast, transforming growth factor beta 1, which is thought to have an important role in the control of growth in colonic epithelium, did not induce apoptosis. Neither RG/C2 nor PC/JW/FI contain wild-type p53, therefore this tumour-suppressor gene is not required to mediate signals for the induction of apoptosis in colonic tumour cells. Our studies report the induction of apoptosis in colonic tumour cells by the naturally occurring fatty acid sodium butyrate. Since sodium butyrate is produced by bacterial fermentation of dietary fibre, the observation that this fatty acid can induce apoptosis could, in part, explain why a high-fibre diet appears to be protective against colon cancer. Escape from the induction of programmed cell death may be an important event in colorectal carcinogenesis.

Adenoma↗
PubMed1989

A rapid micro method for counting cells "in situ" using a fluorogenic alkaline phosphatase enzyme assay.

A new method has been developed to count cells "in situ", based on a fluorogenic enzyme assay that measures the activity of alkaline phosphatase. Increasing cell number was shown to correlate closely with alkaline phosphatase activity and this relationship did not change with time in culture. The alkaline phosphatase assay (ALP assay) was able to estimate relative cell numbers over a range from about 10(4) to 5 X 10(5) for many cell types, including Hep-2, a derivative of HeLa, several human colorectal cell lines SW1222, SW837, LS174T and HT29, a normal human diploid cell strain MRC5 and a rodent line NIH-3T3. The ALP assay is rapid and efficient, making it a useful method for studying growth assays.

Alkaline Phosphatase↗
PubMed1986

The susceptibility of Werner's syndrome and other human skin fibroblasts to SV40-induced transformation and immortalization.

In attempts to transform and immortalize human cell cultures, skin fibroblasts from normal donors of different ages, from patients with the premature ageing diseases Werner's syndrome (WS) and progeria (PR), and from donors with the cancer-prone diseases ataxia telangiectasia (AT), Bloom's syndrome (BS) and Fanconi's anaemia (FA), were infected with SV40 virus and their growth monitored thereafter. Lesch-Nyhan (LN) fibroblasts were also infected. SV40-infected cultures from two normal and from WS, AT and LN donors attained a spectrum of transformed properties, high mitotic activity at confluence, presence of T-antigen, anchorage independence and altered morphology. Most of these pretransformed cultures died in the crisis period. However, two cultures from the WS and LN patients survived the crisis period and have now been grown to more than 200 passages. For the LN culture the crisis period was at least 200 days. Both permanent lines retain the properties of pretransformed cells, but differ in their modal chromosome number and ability to grow in methionine-free medium. It can be concluded from these experiments that transformation by SV40 to permanent lines is a rare event in human skin fibroblasts, even when these cells were taken from patients predisposed to form cancers.

Age Factors↗
PubMed1983

Limited and unlimited growth of SV40-transformed cells from human diploid MRC-5 fibroblasts.

Human foetal lung strain, MRC-5, was treated with simian virus 40 and cultures were obtained that had many of the properties of transformed populations. In 10 experiments, only two produced permanent lines, designated MRC-5V1 and MRC-5V2, which have grown to passage 750 and 650, respectively. In all cases, the SV40-treated cultures acquired many of the features of transformation, including production of T-antigen, loss of contact-inhibition, and ability to grow in low concentrations of serum. The presence or absence of other transformed characteristics, such as altered morphology, abnormal karyotype or ability to grow in soft agar, can be used to distinguish between individual newly infected cultures. However, the cells invariably entered a period of slow growth, or crisis, and in eight experiments the cultures subsequently died without the emergence of a permanent line. The report that late-passage diploid cultures ae more easily transformed to permanent lines than young cultures has not been confirmed. MRC-5V1 initially had a sub-diploid chromosome number, but during serial passaging this gradually increased. MRC-5V2, which has a more extreme transformed phenotype than MRC-5V1, had a hyper-diploid chromosome number, which also increased during long-term growth. MRC-5V1 became polymorphic for glucose-6-phosphate dehydrogenase, as judged by the heat-lability and electrophoretic mobility of the enzyme. Fusions between MRC-5V1 and Lesch-Nyhan fibroblasts yielded hybrids with a limited lifespan, and certain sub-lines of MRC-5V1 also slowed down, exhibited characteristic signs of senescence and ceased to grow.

Bromodeoxyuridine↗
PubMed1981

Cellular aging: further evidence for the commitment theory.

A large, transient reduction in the population size of human fibroblasts in early passages significantly increases the variability of the life-spans of cultures in comparison to control cultures, as predicted by the commitment theory of cellular aging. The theory also predicts that a constant population of noncycling cells will appear in the later part of the culture life-span. This was confirmed by labeling the cells in culture with tritiated thymidine.

Cell Division↗
PubMed1977

Testing the commitment theory of cellular aging.

The commitment theory may explain both the finite lifespan of diploid fibroblasts and the apparent immortality of transformed lines. Potentially immortal cells are assumed on division to generate with some fixed probability cells committed to senesce after a specific number of divisions. During the period between commitment and senescence, cells are assumed to maintain normal growth so that the uncommitted cells are diluted by committed ones and may ultimately be lost in subculturing. A number of predictions of this model are described and experiments strongly supporting the theory are reported. We conclude that the limited growth of diploid fibroblasts is, in effect, an artifact of normal culturing procedures.

Cell Cycle↗
PubMed1977

Age changes of the pattern of F1 histone subfractions in rat liver and spleen chromatin.

The polyacrylamide gel electrophoresis of total histone extracted from young (3 months) and old (26-27 months) rat tissues does not show age-related differences in the pattern of the main five histones. However, when the lysine-rich F1 histone was extracted from chromatin separately by perchloric acid and purified by Biogel P-60 column chromatography from chromatin of rat liver and spleen, the polyacrylamide gel electrophoresis did show obvious age changes in the distribution of F1, F1o, and F1(00) subfractions. In liver chromatin the increase of rat-specific methionine-containing subfraction (F1met.) was also found.

Aging↗
PubMedDate not supplied

A proton magnetic resonance spectroscopy study of aging and transformed human fibroblasts.

Proton magnetic resonance spectroscopy (1H MRS) has been used to monitor changes occurring during aging and transformation in human lung fibroblasts. Aging was studied in MRC-5 cells from nonsenescent (early passage) to presenescent (late passage) and senescence. Nonsenescent cells infected with SV40 virus (pretransformed) were monitored through crisis and subsequent immortalization. Aging changes were observed with one- and two-dimensional MR spectra. Cholesterol and lipid resonances were significantly increased from nonsenescent cultures to senescence. These changes could be caused by chemical or structural changes in the plasma membrane or in intracellular lipid pools. In contrast, choline levels rose from nonsenescent to presenescent cells but at senescence dropped to that of nonsenescent cells. Increased choline levels are often associated with increased cellular proliferation. After SV40 infection of MRC-5 cells there was an increase of cholesterol and lipid levels that peaked at crisis. Newly immortalized cells exhibited a drop in cholesterol and lipid to nonsenescent cell levels, but these rose again in established immortalized cells. In contrast to presensescent cultures, the levels of choline gradually increased from pretransformed to crisis phase but still continued to rise after immortalization. Thus, 1H MRS illustrates similarities in lipid behavior at senescence and crisis, whereas the choline levels are different.

Cell Line, Transformed↗