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T T Puck

Publications and source records attributed to T T Puck.

At least 37 records · Page 2Linked to original sources
PubMed1990

Gene regulation in reverse transformation: cyclic AMP-induced actin homolog in CHO cells.

Reverse transformation (RT) presents a challenge in understanding of the role of protein-genome interaction in regulating gene expression in normal and transformed cells. Early during RT of CHO-K1 cells by cyclic AMP a new protein, mol wt = 43,000 and pI = 5.3 +/- 0.2, was rapidly and specifically induced. This cAMP-induced protein (CIP) is a phosphorylated actin homolog. Induction required new protein synthesis. Actinomycin D treatment failed to inhibit CIP induction, suggesting the existence of an untranslated or sequestered mRNA in untreated cells. Expression of CIP was not dependent upon cell shape or cytoskeletal integrity as are other steps in RT. CIP was detectable only in cAMP-treated cells, whether transformed or nontransformed, and cAMP treatment inhibited growth of both cell types. CIP was associated with soluble cell fractions and not with F-actin. We propose that CIP plays an early role in RT, that is necessary but not sufficient for the complete RT process, and that it participates in the cAMP signaling pathway of cells through changes in the cytoskeleton. This pathway inhibits cell growth as required in the differentiated phenotype. A molecular model is presented for the RT reaction in CHO-K1, which also explains cAMP effects on transformed cells such as the S49 lymphoma and other malignancies.

Actins↗
PubMed1990

The spatial distribution of exposed nuclear DNA in normal, cancer, and reverse-transformed cells.

The malignant CHO-K1 cell is reverse-transformed by cAMP, regaining the phenotype of a normal fibroblast. During this reaction, much of its DNA re-acquires sensitivity to hydrolysis by DNase I in a way characteristic of the normal fibroblast. Exposed DNA forms a rim about the nucleus in both the normal and reverse-transformed cell but not in the malignant CHO-K1. Reacquisition of the nuclear rim requires an organized cytoskeleton. Sequestered DNA forms families of different degrees of sequestration. In accordance with previous theoretical developments it is proposed that (i) genes specific to a given differentiation state are stored in the nuclear rim, whereas genes specific to other states are sequestered within the nucleus; (ii) only exposed genes are active, and their activity is modulated by regulatory molecules in the fluid medium; (iii) exposure and sequestration are regulated by cytoskeletal and nuclear protein structures; (iv) in at least several types of cancer the regulatory defect lies in the genome exposure process so that the specific DNA sequences and their associated growth regulatory loci have been transferred from the exposed to the sequestered condition with consequent loss of the nuclear rim of exposed DNA. The methodology described should be generally applicable to examining the accessibility state of subsets of DNA during various physiological modulations of cell function.

1-Methyl-3-isobutylxanthine↗
PubMed1990

Neuropeptide stimulation of calcium flux in human lung cancer cells: delineation of alternative pathways.

Calcium ion flux following the administration of a series of neuropeptides, N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate, and serum was monitored by flow cytometry in selected lung and breast cancer cell lines and Chinese hamster ovary cell line CHO-K1. Calcium ion flux was monitored in individual cells by flow cytometry using the indicator indo-1 AM. Five groups of neuropeptides produced calcium flux changes in lung cancer cell lines and CHO-K1 cells but not in breast cancer cells. The peak increase in free calcium was reached within 10 sec of peptide administration and declined to resting levels in 70-120 sec. When two or more members of the same group were administered simultaneously, calcium flux changes were identical to that produced by each single peptide. When two or more members of different groups were administered simultaneously, an increased calcium release occurred. When identical peptides or peptides from the same group were administered sequentially after the return of calcium concentrations to resting values, no calcium flux resulted from the second peptide. When peptides from different active groups were administered sequentially, a new calcium flux occurred after each peptide. These data are interpreted to mean that members of each active group of peptides trigger a different calcium flux pathway. Thus, many such pathways and different metabolic states exist within the cell. Elucidation of calcium flux pathways in normal and cancer cells may lead to greater understanding of the nature of the malignant defect.

Amino Acid Sequence↗
PubMed1989

A nuclear protein associated with human cancer cells binds preferentially to a human repetitive DNA sequence.

A protein (Rp66) of 66 kDa was shown by DNA-binding protein blot assay to bind to a human repetitive DNA sequence (low-repeat sequence; LRS) in each of 10 transformed human cell lines examined. This protein-DNA interaction was not observed in 11 normal human cell cultures or in the Chinese hamster cell line CHO-K1. Gel retardation assay confirmed the specificity of the protein-DNA binding between Rp66 and LRS. In a histiocytic lymphoma human cell line, U937, that can be induced to differentiate in the presence of phorbol ester, this binding disappeared after cell differentiation. These together with other results cited suggest a regulatory role for these repetitive sequences in the human genome, with particular application to cancer.

Animals↗
PubMed1989

Involvement of vimentin in the reverse transformation reaction.

An organized cytoskeleton is required for the cAMP-induced reverse transformation reaction in CHO-K1 cells. In the course of the reaction a considerable fraction of the genome changes its nuclease sensitivity. The current paper presents the following evidence that cAMP-induced phosphorylation of vimentin is an early step in this reaction complex. (i) Vimentin is only slightly phosphorylated in transformed CHO-K1 cells but is heavily phosphorylated in normal fibroblasts. (ii) cAMP addition almost triples the vimentin phosphorylation of CHO-K1 cells but does not change that of normal cells. (iii) Vimentin phosphorylation is one of the earliest phenomena to occur after addition of cAMP to CHO-K1 cells, preceding the cell-stretching reaction and other manifestations of reverse transformation. (iv) Indirect immunofluorescence experiments demonstrate that vimentin appears as a condensed mass in transformed CHO-K1 cells but cAMP addition restores the filamentous structure characteristic of the normal fibroblast. (v) Other transformed cells unresponsive to reverse transformation by cAMP failed to demonstrate increased phosphorylation of vimentin on treatment with cAMP. These results support the proposed scheme that phosphorylation of cytoskeletal elements initiates a large-scale genetic regulatory action in which a substantial change in the spectrum of genome exposure and sequestration occurs. A function for intermediate filaments in reverse transformation is implied.

Animals↗
PubMed1988

Analysis of mutant frequency curves and survival curves applied to the AL hybrid cell system.

A model is presented for the statistical analysis of survival curves and mutant frequency curves for a hybrid cell system. The derivation of the model is given in the Appendix, and depends on simple assumptions about the distribution of insults, their repair, and the loss of a marker that is not rescued. A single formula (5) is found which relates a survival curve to the mutant frequency curve, i. e., the response curve for production of mutants per 10(5) survivors induced by a mutagen. The analysis is applied to loss of the a1 gene in AL-J1 hybrid cells submitted to Cesium gamma-rays. Previous experimental data using X-rays was reported by Waldren et al. (1986: Proc. Natl. Acad. Sci, USA 83, 4839.) Also, a derived formula (10), which predicts the probability that in a surviving cell a marker is lost and not rescued, will form the basis for testing the validity of the model in the future using new experimental data.

Analysis of Variance↗
PubMed1988

Specificity of the cAMP-induced gene exposure reaction in CHO cells.

Previous studies demonstrated that in the transformed CHO (Chinese hamster ovary) cell a substantial part of the genome behaves as though its genes are sequestered from effective contact with soluble constituents of the intracellular fluid. The reverse transformation reaction, initiated by cAMP derivatives, causes this cell to regain the morphology, growth regulation, surface characteristics, and sensitivity of its DNA to digestion by DNase I that are characteristic of normal fibroblasts. In this paper we show that this action of cAMP is gene specific. In examination of 47 different genetic loci, some, like ribosomal RNA genes, are found to be sensitive to DNase I hydrolysis both in the absence and in the presence of cAMP; some are resistant under both conditions; and some are resistant in the untreated cell but become sensitive after cAMP treatment. Unlike other gene exposure reactions, which are irreversible and connected with differentiation phenomena, that produced by cAMP is readily reversed when the reagent is removed. A sequence of events is observed after cAMP treatment, the first of which is reorganization of the cytoskeleton. Afterwards, metabolic changes occur over periods as long as 72 hr. The cAMP-induced cytoskeleton-mediated gene exposure reaction appears to be an important genetic regulatory mechanism in mammalian cells and to have special implications for cancer.

Animals↗
PubMed1987

Apparatus for direct counting of beta-rays from two-dimensional protein gels: measurement of changes in protein synthesis due to changes in density of Chinese hamster ovary cells.

A method is described for scanning two-dimensional protein gels that utilizes direct counting of beta-rays instead of autoradiography. The methodology is compared with autoradiographic results and data are presented demonstrating changed patterns of protein synthesis accompanying changes in cell density. The method is rapid and permits identification of differences in protein abundance of approximately 10% for a substantial fraction of the more prominent proteins. A modulation effect of greater than 5 standard deviations is shown to occur for an appreciable number of the proteins that accompany the inhibition of cell growth due to contact inhibition. The method promises application to a variety of biochemical and genetic problems designed to delineate changes in protein synthesis accompanying changes in genome, molecular environment, history, and state of differentiation of the cell populations studied.

Animals↗
PubMed1987

The progesterone receptor gene maps to human chromosome band 11q13, the site of the mammary oncogene int-2.

Progesterone is involved in the development and progression of breast cancers, and progesterone receptors (PR) are important markers of hormone dependence and disease prognosis. We have used a human PR cDNA probe, genomic DNA blotting of a series of Chinese hamster-human cell hybrids, and in situ hybridization to map the human PR gene to chromosome 11, band q13. This band also contains the human homolog of the mouse mammary tumor virus integration site, int-2, which surrounds a protooncogene thought to be involved in the development of murine mammary cancers. That these two genes share the same chromosomal location raises important questions about their possible linkage and about the relationship between the mammary-specific oncogene and the steroid hormone in the development, growth, and hormone dependence of human breast cancers.

Animals↗
PubMed1986

Measurement of low levels of x-ray mutagenesis in relation to human disease.

We previously demonstrated that conventional methods for measurement of mutagenesis in mammalian cells are subject to serious error that causes underestimation of environmental contributions to cancer and genetic disease. This error has been corrected by use of somatic cell hybrids containing a single human chromosome on which the marker genes are carried and by using doses of mutagenic agents so low that little cell killing occurs. This method permits direct measurement of the effects of low doses of radiation and other mutagens without resort to the controversial extrapolation procedure customarily used to estimate effects of doses in the neighborhood of actual human exposures. The new data demonstrate that the true mutagenesis efficiency at the low doses of ionizing radiation that approximate human exposures is more than 200 times greater than those obtained with conventional methods. This methodology also permits evaluation of localized mutations, large and small chromosomal deletions, and nondisjunctional processes and can be used for mutagens that need metabolic activation as well as for cooperatively acting agents. The two opposing classical views that in mammalian cells extrapolation to low doses of x-radiation is linear, on the one hand, or involves a threshold, on the other, are both demonstrated to be incorrect at least for the conditions here considered. The actual curve exhibits a downward concavity so that the mutational efficiency is maximal at low doses. These data may have important implications for human health.

Animals↗
PubMed1984

Cytoskeletal involvement in cAMP-induced sensitization of chromatin to nuclease digestion in transformed Chinese hamster ovary K1 cells.

The cAMP-induced reverse transformation of CHO-K1 cells, which restores fibroblastic morphology, normal nuclear structure, specific membrane structures and biochemical activities, and cell growth regulation, also restores the sensitivity of nuclear chromatin digestion by DNase I to that resembling the normal fibroblast. All of these aspects of the reverse-transformation reaction require integrity of the cytoskeleton. The nuclease-sensitivity effect is achieved only when the entire cell rather than the isolated nucleus is incubated with cAMP derivatives, indicating linkage between the cytoskeleton and nuclear components. Evidence is presented to show that the DNA sensitization to digestion involves interaction between DNA and other chromatin components and affects different regions of the genome in specific ways. Normal fibroblasts display greater endogenous nuclease activity than the transformed cell. The data are interpreted in terms of a genetic regulatory system extending from the membrane to the nucleus and utilizing the cAMP-induced cytoskeleton.

Animals↗
PubMed1984

Localization of the structural gene for human apolipoprotein A-I on the long arm of human chromosome 11.

Apolipoprotein A-I (apo A-I), the major apolipoprotein in human high density lipoproteins, is involved in the disease atherosclerosis. Cloned apo A-I cDNA (pA1-3) was used as a probe in chromosome mapping studies to detect the human apo A-I structural gene sequence in human-Chinese hamster cell hybrids. Southern blot analysis of 13 hybrids localized the gene to human chromosome 11. Confirmation of the chromosomal assignment was obtained by analysis of a hybrid (J1) containing a single human chromosome, no. 11. Regional mapping was achieved by using deletion subclones of J1 that localized the human apo A-I structural gene to the region 11q13 leads to qter. Since the human apolipoprotein C-III (apo C-III) structural gene is closely linked to apo A-I, it can be assigned to the same region on the long arm of chromosome 11. By extension of methods previously described, it now appears possible to carry out fine-structure analysis of this and related gene regions on chromosome 11 and to study the biochemical concomitants of these genes and of genes on other chromosomes for analysis of their role in atherosclerosis.

Animals↗
PubMed1983

Resistance of Chinese hamster ovary cell chromatin to endonuclease digestion. I. Reversal by cAMP.

A portion of the DNA within intact nuclei of a spontaneously transformed Chinese hamster ovary cell line (CHO-Kl) is relatively resistant to digestion by pancreatic deoxyribonuclease, as compared to nuclei from primary cultures of Chinese hamster ovary fibroblasts. Treatment of CHO-Kl cells with derivatives of 3',5' cyclic AMP (cAMP) under conditions which effect the reverse transformation (RT) of these cells, results in restoration of the increased sensitivity of their DNA to hydrolysis by pancreatic deoxyribonuclease, to the level characteristic of an untransformed, morphologically normal Chinese hamster fibroblast cell line. Dibutyryl (db-)cAMP and 8-bromo-cAMP (Br-cAMP) yielded similar results. The cAMP derivatives employed had no effect on the normal fibroblasts.

8-Bromo Cyclic Adenosine Monophosphate↗