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Biomedical subjects

J E Sisken

Publications and source records attributed to J E Sisken.

At least 19 recordsLinked to original sources
PubMed1992

Cytokinesis is more rapid in Ha-T24-ras transfected rat embryo fibroblasts than in non-transfected control cells.

It has long been known that neoplastic cells are characterized by increases in cell motility. Earlier studies from this laboratory indicated that mitotic events were also altered in many tumor and experimentally transformed cells and that this included increases in metaphase duration and a reduction in the duration of cytokinesis. The studies presented in this paper were done to determine whether or not transfection of normal rat embryo fibroblasts by the Ha-T24-ras oncogene could also produce such alterations in mitotic events. The results obtained with the use of time lapse video microscopy indicate that neither the duration of metaphase nor the rate of chromosome movement during anaphase was altered but that the rate of furrow progression during cytokinesis occurred at a significantly more rapid rate. Thus, the cellular alterations induced by transfection with Ha-T24-ras accelerate microfilament-dependent cytokinetic furrowing without significant effects on microtubule-dependent mitotic events. One of several possible mechanisms that could account for these observations involves a down regulation of protein kinase C which has been reported to occur in many neoplastic cells including those transformed by ras. Such a hypothesis could also have broader implications because it may be applicable to the increase in motility and metastatic activity generally observed in transformed cells.

Animals↗
PubMed1991

Sensitivity of cultured human embryonic cerebral cortical neurons to excitatory amino acid-induced calcium influx and neurotoxicity.

Although there has been a large body of literature from animal studies concerning neuronal excitatory amino acid (EAA) receptors and their possible roles in brain development, function, and pathology, essentially no direct information on actions of EAAs in humans has previously been available. We now report on experiments in cell cultured human embryonic cerebral cortical neurons which directly addressed the actions of EAAs in the developing human brain. In cultures established from 14-week fetuses, neurons were insensitive to glutamate neurotoxicity during the first 30 days in culture. After 30 days in culture increasingly more neurons became vulnerable to glutamate acting at the N-methyl-D-aspartate and kainate type receptors. The development of calcium responses to glutamate (as measured with the calcium indicator dye fura-2) preceded sensitivity to excitotoxicity by several weeks in the human neurons. Glutamate-induced rises in intracellular calcium and neurotoxicity developed much more rapidly in rat cortical neurons. Studies of dynamic aspects of calcium responses to calcium ionophore A23187 in human and rat cortical neurons demonstrated a direct relation between calcium buffering ability and resistance to EAA neurotoxicity. Interestingly, the human neurons were better able to buffer a calcium load than were rat neurons, suggesting that species-specific and/or developmental stage-specific differences in calcium-buffering systems are likely to play roles in determining neuronal vulnerability to EAAs. These initial observations indicate that human cortical neurons become sensitive to EAAs during the prenatal period, and suggest that EAAs may play important roles in both normal human brain development and neurodegenerative processes.

Amino Acids↗
PubMed1991

Roberts syndrome with normal cell division.

Roberts-SC phocomelia syndrome (RS) is an autosomal recessive disorder of symmetric limb defects, craniofacial abnormalities, pre- and postnatal growth retardation, and mental retardation. Patients with RS have been reported to have premature separation of heterochromatin of many chromosomes and abnormalities in the cell-division cycle. We report an infant whose clinical and radiologic findings resemble those of RS but who lacks the cytogenetic and cell division abnormalities reported in RS. This patient may represent a variant of RS or a new syndrome.

Abnormalities, Multiple↗
PubMed1987

Transfection of mammalian cells with plasmid DNA by scrape loading and sonication loading.

Scrape loading and sonication loading are two recently described methods of introducing macromolecules into living cells. We have tested the efficacy of these methods for transfection of mammalian cells with exogenous DNA, using selection systems based either on resistance to the drug G418 (Geneticin) or on acquisition of the ability to utilize the salvage pathway of pyrimidine biosynthesis. These loading methods can be employed to generate cell lines that express the gene product of the transfected DNA molecules both transiently and stably. Optimal transfection is observed when the DNA is added to cells in physiological saline lacking divalent cations and containing K+ in place of Na+. DNA molecules 7.1 to 30 kilobases long have been introduced by the scrape loading procedure. In addition, the scrape loading procedure has been employed for cotransfection and subsequent expression of nonselectable genes encoded on DNA molecules added in a mixture with DNA molecules whose expression is selected. Cell lines expressing oncogenes or proteins that are important for regulation of cell growth and division have been obtained by this procedure. The scrape loading procedure is also useful for studies of the cellular changes that occur upon expression of an exogenous gene. As many as 80% of cells scrape loaded with the plasmid pC6, which encodes the simian virus 40 large tumor antigen, contained this protein in the nucleus between 1 and 5 days after transfection. Thus, scrape loading and sonication loading are simple, economical, and reproducible methods for introduction of DNA molecules into adherent and nonadherent cells, and these methods may be useful in the future for experimentation at both fundamental and applied levels.

Animals↗
PubMed1986

The study of fluorescent probes by quantitative video intensification microscopy (QVIM).

In this paper we describe a system for the quantitation and display of fluorescence at the cellular level. It uses a low light level video camera which is interfaced to a fluorescence microscope and to a microprocessor-controlled video digitizing system. With the use of a light pen entry system one can specify areas of the field for measurement. The data obtainable are the area and perimeter of the delimited zone, the distribution of pixel intensities within this zone over a 16-level gray scale, and a value for total fluorescence intensity. Statistical outputs for repeated measurements are also obtained. The system responds linearly to light input, has a high degree of reproducibility, and provides good spatial resolution. Using the DNA-specific dye, Hoechst 33248, in diploid fibroblasts as test material, the system is shown to be able to reproduce expected distributions for amounts of DNA per cell. The capabilities and advantages of pseudocolor display are also demonstrated. We conclude that, in conjunction with appropriate fluorescent probes, systems such as the one described make it possible to do quantitative histochemistry of living cells and to measure substances not previously amenable to study.

DNA↗
PubMed1985

Differential sensitivity of metaphase to diamide and ouabain in HeLa cells.

We have examined the effects of diamide, an oxidizer of glutathione, on the progress of HeLa cells through the cell cycle. At concentrations which do not significantly alter generation time, anaphase or cytokinesis, diamide causes a two-fold increase in the duration of metaphase. At 3 X 10(-8) M, ouabain also prolongs metaphase without effect on anaphase or cytokinesis, though with a different time course. The data suggest that the metaphase stage of mitosis is particularly sensitive to alterations in both sulfhydryl groups and Na+ levels but that the effects of diamide are probably not primarily due to the oxidation of sulfhydryl groups of the Na+/K+-ATPase.

Azo Compounds↗
PubMed1985

Alterations in metaphase durations in cells derived from human tumours.

With the use of time-lapse cinemicrography, we previously found that metaphase durations were significantly prolonged in SV40-transformed human fibroblasts when compared to untransformed controls. This was consistent with some earlier reports and suggested that prolonged metaphases could account for high metaphase/prophase ratios and possibly, in part, for increased mitotic indices seen in advanced tumours. However, there are inconsistencies in the literature and no comparable data available from malignant carcinomas. Presented in this paper are data from two cervical dysplasias, two cases of carcinoma in situ, nine malignant carcinomas and several other types of human cells. The results show that mean metaphase durations were prolonged in cells derived from most of the carcinomas but not from the other cell types. On the other hand, cytokinesis appears to progress more rapidly than normal in most of the tumour-derived cells. These and other findings indicate that the changes are a result of some metabolic alteration common to many but not all tumour cells. For reasons presented, we suggest as a working hypothesis that the alterations may be due to changes in calcium regulation, possibly resulting from alterations in mitochondrial metabolism.

Cell Line↗
PubMed1985

The alteration of mitotic events by ionophore A23187 and carbonyl cyanide n-chlorophenylhydrazone.

A large quantity of published work indicates that calcium ions may be involved in the regulation of mitotic events and recent reports suggest that the onset of chromosome movement is dependent upon a transient increase in free cytosolic calcium ions. In this paper we examine the effects of two agents known to perturb intracellular calcium pools on mitosis in HeLa cells. These were the calcium-selective ionophore A23187 and carbonyl cyanide n-chlorophenylhydrazone (CCCP), which is a protonophoric inhibitor of oxidative phosphorylation. Owing to a stimulation of glycolysis, the latter agent does not decrease intracellular ATP in HeLa but does cause mitochondria to release calcium ions. Our data show that, at low concentrations, both agents prolong metaphase but differ in their effects on anaphase and cytokinesis. Studies with chlorotetracycline, a commonly used probe for membrane-associated calcium, verify that these agents do affect calcium pools under the conditions of our experiments. The data presented are consistent with the idea that increased cytosolic calcium levels can directly or indirectly affect mitotic events but, contrary to other suggestions, cause a prolongation of metaphase, i.e. they delay the onset of chromosome movement.

Calcimycin↗
PubMed1985

Studies on the role of Ca++ in cell division with the use of fluorescent probes and quantitative video intensification microscopy.

It is clear that QVIM systems, when combined with appropriate fluorescent probes can be utilized to perform quantitative cytochemical studies on living and fixed cells. They also have the potential to facilitate studies of substances which like Ca++ are not easily studied by other means. The preliminary studies we have described support the idea that calcium ions and calcium transport enzymes may indeed play important roles in cell division and indicate that the tools we have at hand should help us further our understanding of the mitotic process.

Animals↗
PubMed1979

On the duration of mitotic stages in senescing human fibroblasts in culture.

The duration of three mitotic periods was measured by time-lapse cinemicrography in a line of normal human fibroblasts which displays the phenomenon of limited growth potential in vitro. It was found that the durations of metaphase and anaphase did not change as a function of passage level but that there was a statistically significant increase in the duration of cytokinesis. It was also found that there was no progressive increase in the variance of these periods between early and late passage cells. The data suggest that for those late passage cells which still retain the capacity to divide, the cellular mechanisms responsible for the synthesis of functional proteins, energy regulation and at least some membrane functions appear to be working normally.

Anaphase↗
PubMed1979

The effects of nicotine on cell division of HeLa cells.

A time lapse cinemicrographic study shows that, at low concentrations, nicotine can speed up cytokinesis and, at high concentrations, prolong the duration of metaphase in HeLa cells. Based upon the known effects of the alkaloid in other systems, it appears that it may stimulate microfilament activity and inhibit microtubule function by increasing the level of cytosolic calcium ions.

Cell Division↗