Irradiated HeLa Cells Research Articles

New factors relevant to polyamine levels in cancer patients.

Results presented show that the polyamines putrescine, spermidine and spermine are degraded in human and bovine serum. Degradation of putrescine in human serum is inhibited by the anti-cancer drugs (±)-1,2 bis (3,5-dioxopiperazin-1-yl) propane (I.C.R.F.-159) (Razoxane I.C.I.), Doxorubicin HCl (Adriamycin, Pharmitalia) and 5-fluorouracil so that polyamine levels are higher than in controls. In calf serum degradation of spermidine is accompanied by formation of putrescine. Irradiated He-La cells give rise to cultures with higher polyamine levels than cultures from non-irradiated cells. Urine polyamine levels of cancer patients receiving drugs and radiation were not well correlated to the clinical progress of their disease. Accordingly, changes in polyamine levels of cancer patients may arise from therapy independently of tumour response and may be unreliable for assessment of tumour state and response to treatment.

Observations of the First Postirradiation Division of HeLa Cells Following Continuous or Fractionated Exposure to γ Rays

The first postirradiation division of synchronized S3 HeLa cells was studied using both continuous and fractionated irradiation treatments. Synchronized HeLa cells continuously irradiated at a dose rate of 37 rad/hr eventually accumulate in mitosis. If the continous irradiation is stopped before the cells enter G2 or even after they have progressed for a limited time into the G2 arrest that develops, very little subsequent accumulation of cells in mitosis occurs. If they progress for a longer time into the G2 arrest, then some mitotic accumulation does occur after the irradiation is stopped. When synchronized cells were allowed to progress through G1 and S before the irradiation was started, very little cell division occurred during subsequent continuous irradiation and extensive mitotic accumulation was observed. Thus, for continous irradiation of HeLa cells, the dose received by a cell during G2 or a G2 delay apparently determines whether it will be able to divide if it reaches mitosis. Arguing against ...

Modification of Radiation Response in HeLa Cells by Misonidazole

The hypoxic radiosensitizer misonidazole decreases survival in dense cultures of HeLa cells irradiated with gamma rays in a non-dose modifying fashion. Survival curves of treated hypoxic cells display a much larger extrapolation number than untreated cells. In oxygenated randomly dividing cells, drug treatment has an effect opposite to that in hypoxic cells, increasing survival. In cultures initiated from mitotic cells and irradiated soon afterwards, a smaller sensitization under hypoxia and no increase in survival of oxygenated cells was observed. It was concluded that metabolic as well as radiochemical events take place in misonidazole-treated and then irradiated HeLa cells which modify survival.

The influence of inhibitors on dimer removal and repair of single-strand breaks in normal and bromodeoxyuridine substituted DNA of HeLa cells

The elimination of cyclobutane pyrimidine dimers from the nuclear DNA of ultraviolet irradiated HeLa cells has been examined by means of chromatography and immunoautoradiography. The extent and duration of the process was similar when dimers were assayed by both methods, proving that the antisera recognized pyrimidine dimers. The rate of dimer excision did not differ through the cell cycle with the exception of mitosis during which no dimers were removed. Dimer excision is a relatively fast process which is terminated within a few hours, but it leaves many dimers in the DNA. Excision is depressed by inhibitors of semiconservative DNA synthesis that affect the DNA precursor pool or DNA polymerases. Cells whose DNA is partly substituted with bromodeoxyuridine instead of thymidine, repair single-strand breaks and remove dimers at the same rate but to different extents. On the other hand, inhibitors limit repair of breaks and removal of dimers to the same degree suggesting that the repair of the two types of lesion is coordinated.

The inhibition of repair in UV irradiated human cells

We have used three different assay procedures to determine the effects of hydroxyurea on excision repair in UV-irradiated HeLa cells. The results were as follows. (a) At the cytological level, incubation of UV-irradiated metaphase cells with hydroxyurea caused chromosome decondensation. (b) Using a modified alkaline sucrose gradient sedimentation technique involving minimal lysis before centrifugation, we found a marked retardation in the sedimentation of DNA from UV-irradiated cells incubated for a short period with hydroxyurea. (c) The effect of hydroxyurea on the incorporation of[3H]thymidine by UV-irradiated G1 cells was found to depend on the concentration of thymidine present in the medium. Normal primary human cells resemble HeLa cells in the response of chromosomes and DNA to UV plus hydroxyurea. Xeroderma pigmentosum cells, deficient in excision repair, are not sensitive to hydroxyurea in our assays. Chromosome decondensation and retarded DNA sedimentation occur also after incubation of irradiated HeLa cells with deoxyadenosine, but not thymidine, at concentrations which inhibit semiconservative DNA synthesis. The effects of hydroxyurea or deoxyadenosine on chromosomes and DNA are not seen if all four deoxyribonucleoside precursors of DNA are supplied exogenously. These results point to an inhibition of repair DNA synthesis by hydroxyurea (or deoxyadenosine), at the level of the supply of DNA precursors, i.e. in the same way that these agents inhibit semiconservative DNA synthesis. In the presence of these inhibitors, single-strand gaps accumulate in the DNA.

The Enhanced Killing of Irradiated HeLa Cells in Synchronous Culture by Hyperthermia

Mitotically synchronized cultures of HeLa S-3 cells were subjected to the treatment of radiation (400 rad), hyperthermia (43/sup 0/C), and a combination of both at different phases of the division cycle. The radioresistance was most pronounced in the mid G-1 and late S phases, while thermal resistance was greatest in the early G-1 phase and steadily decreased as cells entered the S phase. Cells in the late S and early G-2 phases were found to be most sensitive to hyperthermia. The sequential treatment of radiation immediately followed by hyperthermia resulted in an enhanced cell killing throughout the cell cycle with a marked synergism occurring in cells in the late S phase. The age-response function of the combined treatment was more similar to that of the thermal age response.

Letter: Formation of sulphydryl peptides ol low molecular weight in HeLa cells after irradiation.

PCMBS (p-chloromercuric-phenyl sulphonic acid), labeled with /sub 203/Hg was used to determine -SH containing peptides formed after irradiation of HeLa cells. Sulphydryl groups of peptides bind firly and specifically with PCMBS and the labeled compound bound to the -SH containing cell components was separated from unbound PCMBS on the basis of molecular size by Sephadex chromatography. Comparison of control and irradiated cell components showed very few qualitative differences, but significant quantitative differences were observed in the amino acid composition of the low-molecular-weight components separated on the G25 column. Very large increases in aspartic and glutamic acids, threonine, serine, and alanine were observed after irradiation. Studies of -SH components separated by G100 Sephadex showed that a redistribution of -SH had occurred after irradiation and that components formed were probably smaller than 5000 in m.w. The use of G25 columns demonstrated that most -SH components had a molecular weight within range of a few hundred, probably 100 to 400. G10 column fractionations confirmed the existence of small di- or tripeptides in addition to cysteine itself. (UK)

Absence of a fractionation effect in irradiated HeLa cells.

SummaryAerated and hypoxic HeLa cells have been exposed to single and split doses of radiation using pulses of fast electrons and conventional X-radiation. With aerated cells the single dose survival curves exhibited a shoulder with both types of radiation, before becoming exponential, but no fractionation effect was observed in the experiments with fast electrons, although one was found with X-rays. For cells under conditions of extreme hypoxia the single-dose survival curves were exponential both with electron pulses and with X-rays, and split-dose experiments showed no fractionation effect.

Intracellular radiation of HeLa cells by tritiated thymidine. I. Lactic acid production and exponential growth.

SummaryThe production of lactic acid by irradiated and unirradiated HeLa cells, grown as monolayer, was investigated. As long as exponential growth was maintained, the lactic acid production was proportional to the increase in cell number, and the production per hour and per cell was constant. When tritiated thymidine was added to the medium, a significant increase of 23 per cent of the lactic acid production per hour and per cell was found for the highest doses. The findings are discussed with regard to the proposed equations and related to the calculation methods of other workers.

Dissociation of histone and DNA synthesis in X-irradiated HeLa cells

Although histone synthesis and DNA synthesis are normally very well coordinated in HeLa cells, their histone synthesis proved relatively resistant to inhibition by ionizing radiation. During the first 24 h after 1 000 R the rate of cellular DNA synthesis progressively fell to small fractions of control values while histone synthesis continued with much less relative reduction. Acrylamide gel electropherograms of the acid soluble nuclear histones synthesized by irradiated HeLa cells were qualitatively normal.

Utilization of host-cell DNA by vaccinia virus replicating in HeLa cells irradiated intranuclearly with tritium.

SUMMARY When HeLa cells were pre-labelled with tritiated thymidine and infected with the wr or ihd strain of vaccinia virus, the host-cell DNA was broken down and reutilized for virus particle DNA biosynthesis. This phenomenon did not occur in LM cells, and was not explained by pseudovirus particle formation. Utilization of host-cell DNA, pre-labelled with [14C]-thymidine did not occur. Therefore, breakdown and re-utilization of host-cell DNA in HeLa cells resulted from intranuclear irradiation of the cells. Under these circumstances there was a markedly increased activity in the cells of an exonuclease with a pH optimum of 9.2, a requirement for magnesium ion, and which used denatured DNA as a substrate. The activity of this enzyme in such cells was directly proportional to the amount of irradiation, measured as the specific activity of the pre-labelled host-cell DNA. Associated with the vaccinia virus particles obtained from intranuclearly irradiated HeLa cells was an alkaline DNase inhibited by magnesium ion.

Temperature dependence of thymidine uptake and ability of thymidine incorporated during unscheduled DNA synthesis in irradiated and unirradiated HeLa cells.

Temperature dependence of thymidine uptake and ability of thymidine incorporated during unscheduled DNA synthesis in irradiated and unirradiated HeLa cells.

Modification of radiation response in synchronized HeLa cells by metabolic inhibitors. I. Effect of an inhibitor of RNA-synthesis, puromycin aminonucleoside.

SummaryTreatment of irradiated HeLa cells by puromycin aminonucleoside (PAN), an inhibitor of ribosomal RNA-synthesis, markedly reduces colony-forming ability. Post-irradiation treatment by PAN in the mitotically synchronized cells shows that the highest potentiating effect of PAN is observed at the end of the S phase, normally the most radioresistant part of the cell cycle, and least effective during G-2 and G-1 phases, in that order.Based on the known mechanism of action of this drug, the possible role of certain metabolic events in radiation response of living cells is discussed.

Fallopian tube and early cleavage in the mouse.

TECHNIQUES have been perfected for the culture in vitro of late two cell mouse ova into blastocysts in simple chemically defined conditions1,2. Attempts to obtain development from earlier stages have failed, unless the ova were cultured in organ cultures of the fallopian tube3,4. These results suggested that the fallopian tube provides some critical contribution necessary for the development of the mouse zygote into the late two cell stage. Nevertheless, it has been a common experience that when the mouse zygote is cultivated independently of the fallopian tube a few cleave to the two cell stage and then cease development5. It has now been shown6, however, that a high percentage of zygotes cleave to two cell stages and then cease development when cultured in Waymouth7 medium supplemented with ATP, deoxynucleosides and a feeder layer of irradiated HeLa cells. These experiments suggest that the initial development of the mouse zygote is dependent on a supply of exogenous factors, additional to those required by later stages.

Restoration of radiation damage examined by the analysis of HeLa cell clones.

This paper reports a small, but significant, increase in the survival of irradiated HeLa cells after treatment with DNA precursors. Clone-size analysis is used to indicate that this may be regarded as a true restoration of radiation damage and not merely a nutritive phenomenon. Single cell survival was estimated by the technique of Puck and Marcus (1956) and involved the usual comparison of the numbers of clones which have attained a minimum size within a given period. This criterion provides evidence of the reproductive integrity of single cells, but it conceals a further criterion of survival, namely the ability of a cell to proliferate at the normal rate. Analysis of HeLa cell clones has shown (Nias, Gilbert, Lajtha and Lange 1965) that a broad spectrum of clone sizes may be found after irradiation, as described for Chinese hamster cells by Sinclair (1964). Such broad clone-size distributions indicate the range of proliferation rates induced by radiation. These rates extend from the maximum control rate down to zero. Those within the control range can now be defined as ' survivors' in the full sense: cells able to proliferate at the control rate. Smaller clones may well attain the 50-cell level, given time, but these exhibit radiation damage, albeit non-lethal damage. If radiation damage is restored by treatment with DNA precursors, such restoration may involve two effects: (a) an increase in the number of cells with reproductive integrity; (b) an increase in the proliferation rate of non-lethally damaged cells. The second effect might be considered to be a growth-promoting phenomenon as distinct from the first which requires an absolute increase in the number of viable cells. The usual clone counting procedure cannot distinguish the two effects, however, since an increase in the proliferation rate or non-lethally damaged cells may produce more 'over-50-cell' clones just as much as would an increase in the absolute number of viable cells. Clone-size analysis allows these two effects to be examined. A difference in clone-size distribution implies either a growth-promoting or a toxic effect (e.g. figure 1). If the clone-size distributions are similar, on the other hand, any increase in the number of 'over-50-cells' clones implies an increase in the number of viable cells. This would represent a true restoration of radiation damage to the reproductive integrity of the cells.

Continuous irradiation of HeLa cells at--196 degrees C.

THE incorporation of a suitable quantity of tritiated water in the growth medium permits the continuous irradiation of cells cultured in vitro. A uniform source of β-radiation is thus provided which can be used in a variety of cell culture techniques. Some observations on the effects of continuous irradiation of HeLa cells grown as a monolayer at 37° C have already been published1,2. This communication reports some results of the continuous irradiation of HeLa cells at −196° C.

Change in chromosome number during continuous irradiation.

A PREVIOUS communication1 reported some quantitative data on the effects of continuous irradiation of HeLa cells grown as a monolayer. The incorporation of a suitable quantity of tritiated water in the growth medium provided a uniform source of radiation. An effect was noted with a dose-rate as low as 30 rads/day, when cells were repeatedly sub-cultured. Cells have now been exposed to such continuous irradiation for more than twelve months and this communication reports changes in the chromosome number of the continuously irradiated cells.

Effect of x-radiation on uptake of P32 into individual nucleotides of HeLa cells.

Separation of 24 acid soluble phosphorus compounds from irradiated and nonirradiated HeLa cells is described. Relative specific activities of 19 of these compounds are given. lrradiation of the cells produced an increase of relative specific activity and in amount of uridine diphosphoacetylglucosamine and a decrease in amount and specific activity of adenosine triphosphate.

Biochemical changes during the formation and growth of giant cells from irradiated HeLa cells

The formation and growth of giant HeLa cells in response to irradiation has been followed by means of colorimetric analyses for nucleic acids and protein and by the incorporation of radioisotopes into protein and deoxyribonucleic acid. Thymidine-2- 14C uptake into cellular deoxyribonucleic acid continued at essentially normal rates for ten days after recovery from irradiation and then declined. The rate of incorporation of glutamic acid- 14C reached a level greater than that in the control cells on a per cell basis, and protein synthesis extended for a number of days beyond the capacity for deoxyribonucleic acid synthesis.