Interaction of hyperthermia and pentamidine in HeLa S-3 cells

Pentamidine is similar to rhodamine-123 in chemical structure and state of electron charge, and rhodamine-123 was previously shown to be a hyperthermic sensitizer under appropriate cell culture conditions. The present experiments were carried out to determine whether pentamidine would potentiate hyperthermic cell killing and, if so, under what cultural conditions. Exposure of HeLa cells to pentamidine (80 microM) up to 4 h was not cytotoxic in culture medium in the presence or absence of glucose at 37 degrees C and pH 7.4. Cells in the glucose-deprived medium became progressively sensitive to killing as temperature and drug concentration were increased. On the other hand, there was a moderate level of enhanced cell killing in the glucose-fed medium at 42 degrees C. The enhanced effects of heat by the drug were most pronounced under alkaline pH of the culture medium. The cell kill under acidic pH was far less than that observed at neutral or alkaline pH; these effects may be a result of decreased cellular uptake of pentamidine. Together with our previous results on rhodamine-123 and glycolytic inhibitors, the present data with pentamidine are consistent with the concept that deprivation of cellular energy increases sensitivity to cell killing by heat.     

Repair of radiation-induced DNA damage in thermotolerant and nonthermotolerant HeLa cells

The effect of heat exposure on the repair of radiation-induced DNA damage which inhibits the ability of nuclear DNA to undergo supercoiling changes was studied using the fluorescent halo assay in thermotolerant and nonthermotolerant (normal) cells. The assay utilizes an intercalating, fluorescent dye to unwind and rewind endogenous DNA supercoils. When HeLa cells are exposed to 17.3 Gy radiation the ability of DNA to be rewound into supercoils is completely inhibited. However, the ability of DNA to rewind is 70% restored by 30 min after irradiation. Both thermotolerant and normal cells exposed to 45 degrees C for 30 min prior to irradiation had a rewinding ability intermediate between control and unheated cells, but there was no restoration of rewinding ability up to 3 h postirradiation. Thus, when irradiation immediately followed heating, there was no difference between thermotolerant and normal cells. However, when various time intervals were imposed between heating and irradiation, a difference in the ability of the cells to recover from heat-induced alterations became apparent. In normal cells after 6 h of postheat incubation the cells' ability to restore DNA supercoiling was approximately the same as that of control cells, while in thermotolerant cells only 2 h was required to repair the ability to restore supercoiling at the same rate. The rate of repair of DNA remained correlated with relative nuclear protein content as measured by fluorescein isothiocyanate staining in both thermotolerant and normal cells, indicating a possible relationship between the two.     

Mitosis and protein synthesis. 3. Organelle relocation during normal and colcemid-arrested M-phase in HeLa S-3 cells

During M-phase, most organelles in HeLa S-3 cells are relocated in the 'cortex' or outer-zone of cytoplasm. Elements of the rough endoplasmic reticulum (rER) and polysome assemblies persist to varying degrees from cell to cell in this zone. Dilatation of rER cisternae becomes prominent in a small percentage of metaphases, and its occurrence and significance is discussed. Remnants of the Golgi apparatus are almost invariably peripheralized. Its cisternal elements are lost in early mitosis, and reappear in late telophase. The inner zone of the protoplasm around the chromosomes loses its associated intermediate filaments and excludes organelles until cytokinesis commences. A rapid repopulation occurs by mid-telophase. The same pattern of zoning is found in cells entering mitosis in the presence of colcemid, but is followed by some repopulation of the inner zone by a small minority of organelles after approximately 2 h of arrest. Centrioles are particularly prone to becoming enmeshed within the 'ball' of entangled c-metaphase chromosomes. An unusually high degree of pairing of cytoplasmic membranes, probably rER elements, also occurs in colcemid-arrested metaphases, which may further contribute to their reduced level of protein synthesis. These have been referred to as 'confronting cisternae' (Ghadially, 1988). The zoning of the cytoplasm may result from nuclear envelope breakdown during mitosis, and is not specifically related to or associated with microtubule redeployment during spindle formation in M-phase. Differences in the extent of zoning in other cell lines are discussed in comparison with HeLa S-3 cells.     

Investigation of the mechanism of protein turnover in HeLa S-3 cells by incubation at elevated temperatures

An apparent paradox relating to the degradation of endogenous proteins in HeLa S-3 cells occurs at 45 degrees C, at which their proteolysis is considerably enhanced in vitro but completely inhibited in vivo. No significant differences in rates of degradation of short-lived (nascent) and long-lived ('existing') proteins synthesised at 37 degrees C were found when chased at temperatures up to 43 degrees C, but at 45 degrees C degradation of both categories was reduced to zero in vivo. Synthesis of protein was suppressed at temperatures above 41 degrees C, being reduced by up to 60% at 43 degrees C. Proteolysis in vitro proceeded 1.6-1.7 times faster at 45 degrees C than at 37 degrees C and neutral pH. Evidence is presented for the involvement of the basal system; the findings both in vivo and in vitro do not seem to implicate the lysosomal system, no firm indication being obtained of its 'induction' at elevated temperatures. The results are discussed in terms of the arrest of intracellular circulation at elevated temperatures, thereby reducing the delivery rate of proteins as substrates of the intracellular basal proteolytic enzyme system to negligible levels (i.e., to the frequency of encounters due solely to the diffusion of protein molecules with the cytoplasm).     

Turnover of nuclear RNA in HeLa cells: evidence for a single ribonucleotide pool

The role of the ribonucleotide pool in measuring turn-over of RNA has been studied. Evidence presented previously has assumed a single pool. The nucleotide base ratios of two different species of nuclear RNA (heterogeneously sedimenting nuclear RNA and ribosomal precursor RNA) are known. We have studied the rate of approach to the equilibrium base ratio established by labeling, either via the salvage pathway or the endogenous route. In either case, normalization of the measured U:C ratios is accomplished by dividing the equilibrium U:C ratio in an isolated species by the measured base ratio (specific U:C ratio). These studies are consistent with the existence of a single ribonucleotide pool.     

A radiation-induced inhibitor of chromosome condensation and nuclear envelope breakdown in HeLa cells.

An in vitro microscopic assay for mitosis-inducing activity in mitotic HeLa cells was developed and used to demonstrate that cells irradiated and arrested in G2 phase of the cell cycle contain an inhibitor of mitosis. This assay system has a number of advantages over other assays including the use of autologous components (HeLa nuclei and mitotic cell extracts) in contrast to the microinjection method with Xenopus oocytes and without the requirements for microinjection expertise and Xenopus oocytes. The radiation-inducible inhibitor was detected at the lowest radiation dose tested (2 Gy) with maximal activity achieved within 30 min after radiation. Inhibitor activity decayed with time after radiation (2 Gy) with no activity detected at 6 h even though the cells remained in G2 phase, suggesting that either synthesis or activation of additional components is necessary for recovery from G2 arrest. The inhibitor activity was not detected in irradiated cells treated with caffeine to induce premature recovery from G2 arrest.     

Interactions between human fibroblasts and HeLa cells in vitro

Affinity toward each other was demonstrated in co-cultures between HeLa cells and fibroblasts originating from human tumor stromal or normal tissues. Both cell types in the mixed cultures (ratio 1:1, 1:2, 2:1) proliferated normally as shown by 3H-thymidine labeling index estimation for up to 48 hr of co-culture. At ratios of fibroblasts: HeLa lower than 1:10, fibroblasts were eventually eliminated after serial passaging. It was shown that 3H-nucleotides could be transferred between heterologous cells in either direction. Contact of cells was essential for this phenomenon. Transfer of the label from HeLa to fibroblasts required a longer interaction time and was evidently lower than the transfer from fibroblasts to HeLa. 3H-thymidine incorporated into the DNA of either cell type could not be transferred from one cell to another. The model provides a means for studying neoplastic X normal (or tumour stromal) cell interactions in vitro.     

Spawning synchrony in Arenicola marina: evidence for sex pheromonal control

Chemical communication systems controlling reproductive behaviour have been shown in a number of marine polychaetes. This study investigated the use of sex pheromones to coordinate spawning behaviour in gravid lugworms (Arenicola marina). Lugworms typically reproduce in the autumn, during low water of spring tides, and often exhibit epidemic spawning. Females release gametes within the burrow whereas males deposit spermatozoa on to the beach surface. The incoming tide dilutes the spermatozoa and transports them to the females'' burrows. Sperm is diluted rapidly and sperm concentrations fall below the minimum required for fertilization within a few minutes. The present investigation establishes the existence of chemical signals synchronizing spawning for the first time in an iteroparous polychaete. The process can be divided into two steps, the induction of gamete release by waterborne chemical cues and burrow irrigation behaviour in females: burrow irrigation representing the means by which spermatozoa are carried to the eggs. In both sexes, the release of gametes can be induced by exposure to sea water into which other individuals had previously spawned. Males also respond to odour compounds from other males. The overall effect of the chemical signals results in synchronized, mass spawning of a population.