Equivalence of pulsed-dose-rate to low-dose-rate irradiation in tumor and normal cell lines

To determine whether different fractionation schemes could simulate low-dose-rate irradiation, ovarian cells of the carcinoma cell lines A2780s (radiosensitive) and A2780cp (radioresistant) and AG1522 normal human fibroblasts were irradiated in vitro using different fraction sizes and intervals between fractions with an overall average dose rate of 0.53 Gy/h. For the resistant cell line, the three fractionation schemes, 0.53 Gy given every hour, 1.1 Gy every 2 h, and 1.6 Gy every 3 h, were equivalent to low dose rate (0.53 Gy/h). Two larger fraction sizes, 2.1 Gy every 4 h and 3.2 Gy every 6 h, resulted in lower survival than that after low-dose-rate irradiation for the resistant cell line, suggesting incomplete repair of radiation damage due to the larger fraction sizes. The survival for the sensitive cell line was lower at small doses, but then it increased until it was equivalent to that after low-dose-rate irradiation for some fractionation schemes. The sensitive cell line showed equivalence only with the 1.6-Gy fraction every 3 h, although 0.53 Gy every 1 h and 1.1 Gy every 2 h showed equivalence at lower doses. This cell line also showed an adaptive response. The normal cell line showed a sensitization to the pulsed-dose-rate schemes compared to low-dose-rate irradiation. These data indicate that the response to pulsed-dose-rate irradiation is dependent on the cell line and that compared to the response to low-dose-rate irradiation, it shows some equivalence with the resistant carcinoma cell line, an adaptive response with the parental carcinoma cell line, and sensitization with the normal cells. Therefore, further evaluation is required before implementing pulsed-dose-rate irradiation in the clinic.     

Repopulation in murine skin after X-ray treatments with multiple fractions per day

The kinetics of repopulation of clonogens in skin after fractionated X-ray exposures was studied in a series of experiments using a top-up design. The feet of mice were exposed to small X-ray doses (1.5 or 2 Gy), given two or three times a day on consecutive days with a minimum interfraction interval of 8 h. A single top-up dose of d(4)-Be neutrons was then given at various intervals after the last X-ray fraction, typically on Days 1,4,8, 15, and 19. The acute skin reaction produced was scored an analyzed by both a standard 23-day averaging and a 7-day averaging procedure. Either method gave similar results and led to the same conclusions. The amount of top-up dose needed to produce a fixed skin reaction was used as a measure of the net effect of the X-ray treatments. This net effect is a result of the initial reduction in skin clonogens due to X rays, and their repopulation before the top-up dose was given. Repopulation was not detected during any of these courses of fractionated treatment, up to an overall time of at least 12 and possibly 16 days. On completion of X-ray schedules lasting 6-16 days, repopulation started 4 days later. In contrast, this delay lengthened to approximately 8 days for shorter overall treatment times of 3-4 days. Once repopulation started, it proceeded rapidly over 11 days so that by 15 days after the cessation of X rays, the skin was restored almost to its normal state with respect to radiosensitivity. The residual damage from Day 15 to Day 19 postirradiation was 3-13% of a full-effect level. The rate of repopulation can be expressed as a clonogen doubling time (Tclon), assuming that an average skin reaction of 1.5 is equivalent to a clonogen surviving fraction of 1.7 x 10(-5). Tclon varied inversely with the amount of initial damage inflicted by the X rays, with the shortest values (1-1.3 days) seen following X-ray doses that gave an initial damage level of 60-80% of full effect. These data are consistent with a hypothesis that damage is "sensed" only 10-12 days after the first X-ray fraction, which provides the stimulus for repopulation of the target cells in the basal layer, the keratinoblasts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of lymphokine-activated killer cell generation by cultured tumor cell lines in vitro

Summary The co-culture of human peripheral blood mononuclear cells (PBMC) with high concentrations of interleukin 2 normally generates lymphokine-activated killer (LAK) cells capable of indiscriminate lysis of tumor targets. However, the addition of certain cell-line-derived tumor cells to the LAK generation cultures within the first 48 h of culture initiation resulted in the suppression of the LAK cytotoxicity measured after 3–4 days of culture. Suppression could be achieved with tumor cell:PBMC ratios as low as 1:50 when tumor cells were derived from melanoma and colorectal cancer (G361, COLO320, HT-29), but suppression was not observed with cells from the breast cancer cell line SKBr3. No suppression of LAK generation was observed with normal epithelial cells from colon or breast, with autologous or allogeneic lymphoblasts, or with allogeneic vascular endothelial cells. Suppression was independent of the removal of adherent cells from PBMC, could not be prevented by indomethacin and was not attributable to interleukin 2 absorption/adsorption by tumor cells. The suppressive activity of some tumor cells could be augmented by preculture in recombinant gamma interferon. Serum-free supernatants from G361, COLO320 and HT-29 (but not SKBr3 or endothelial cells) were also highly suppressive towards the generation of LAK cells. The elaboration by tumor cells of fractors capable of inhibiting LAK generation may partially explain the failure of LAK/interleukin 2 therapy in some experimental and clinical protocols.     

Prolonged hyperthermia eliminates mycoplasma from cultured human and rat brain tumor cell lines

Mycoplasma infection of mammalian cells in culture is a common occurrence that can affect the results of experimental protocols. Current methods of eliminating mycoplasma from cell cultures are usually tedious, time-consuming, and sometimes unsuccessful. In the present study, four cultured brain tumor cell lines (human U-251 MG, U-87 MG, SF-126, and rat 9L) were heavily contaminated with Mycoplasma orale. Heating the cultures to 41 degrees C for at least 96 h eliminated the contamination for up to 7 months, the maximum period of observation. The time chosen to assay for the presence of mycoplasma in cultures was critical: in some cultures heated for less than 96 h that initially appeared to be free of contamination, mycoplasma began to appear after 2 weeks. Heat-treated cells grew at the same rate as unheated control cells. Infected cells were more sensitive to X rays than uncontaminated cells, but the sensitivity reverted to normal after mycoplasma was eliminated by hyperthermia. The heating method does not require a cell cloning procedure or the use of exogenous materials. Treated cell cultures exhibit normal growth and radiation sensitivity, and the technique seems to be reliable and efficient.     

A two compartment model for cell survival after irradiation with ionizing radiation

A theory of cell survival after irradiation has been developed, considering the cell as composed of two compartments with different sensitivities and taking into account recovery phenomena. Expressions are obtained for the probabilities that the cell will be in a survival state or damaged state or will function with reduced efficiency.     

Analysis of cell kinetics using BrdU monoclonal antibody on cultured tumor cells after irradiation and treatment with anti-cancer drugs

Flow cytometry (FCM) permits instantaneous determination of the percentages of cells in various phases of cell cycle using BrdU-PI double staining method, and allowing rapid evaluation of the effects of irradiation and anti-cancer drugs (ACNU, ADR, BLM) on the cell kinetics. In this study, the growth inhibition and changes in the cell kinetics after irradiation and chemotherapy were examined according to the growth curve analysis and BrdU-PI method to evaluate the usefulness of BrdU-PI method for assessment of the effect of the treatments. By the conventional method based on the DNA histogram, accurate determination of S cell fraction was difficult due to overlapping of the DNA contents of G1 cells and early S cells and those of late S cells and G2 cells. BrdU-PI double staining allowed direct differentiation of G1, S, and G2 + M cells, especially between G1-S and S-G2 + M cells. The analysis of cell kinetics using BrdU is advantageous in comparison to the conventional autoradiographic methods because it allows more rapid assay with very high sensitivity. By the present BrdU method, rapid transition to the G1-S phase was observed within 4 hours after exposure to radiation and anti-cancer drugs. This initial G1 arrest induced by irradiation was confirmed for the first time by the present BrdU-PI double staining. The present method is considered to be indispensable for evaluation of the percentage of S cells in the tumor tissue and analysis of cell kinetics after irradiation and chemotherapy against cancer.     

The effect of ascorbic acid on RNA and protein synthesis on two cultured cell lines in vitro

Previous work in this laboratory has thrown some light on the possible mechanism involved in the anti-tumour activity of ascorbic acid (AA). In order to elucidate this mechanism further, the present studies, involving the effect of AA on protein and RNA synthesis, were carried out. The results obtained in this investigation may support the hypothesis previously put forward for the action of AA on cell proliferation.     

Epithelial and fibroblast cell lines cultured from the transgenic BigBlue rat: an in vitro mutagenesis assay

We have isolated, cultured, and immortalised three new BigBlue transgenic rat cell lines for the study of mutation induction in vitro. The two epithelial cell lines, from the mammary gland and oral cavity, were designated BBR/ME and BBR/OE, respectively, and the third is a mammary fibroblast line designated BBR/MFib. We have characterised these cell lines with respect to chromosome number and the expression of some cell-specific antigens. The clonogenic survival and cII transgene mutation induction responses of these three cell lines to N-ethyl-N-nitrosourea (ENU) treatment were determined. Both epithelial cell lines were much more sensitive to ENU toxicity than was the fibroblast cell line. However, all cell lines showed similar ENU dose-dependent increases in mutant frequency. We hope that cell lines such as these will extend the power of the BigBlue assay to in vitro studies.     

Thiamine requirement of two different cultured cell lines of soybean

When supplied with 6-benzyladenine (0.5–5 mg/liter) insteadof thiamine, thiamine-requiring soybean cells (strain TU) couldgrow successively. The effect of cytokinin was much more remarkableat a relatively higher concentration of 2,4-dichlorophenoxyaceticacid (4 mg/liter) than at a lower concentration (0.5 mg/liter).Among calli initiated from soybean hypocotyls on a medium withoutthiamine, the thiamine-nonrequiring variant (strain G) was obtainedincidentally. As this cell line became green in light, it couldbe visually separated from the other necrotic tissues. StrainG cells could grow successively not only without thiamine butalso without phytohormones, auxin and cytokinin. This cell linehad relatively higher amounts of chlorophyll and thiamine, andgrew in rigid, large cell aggregates which differed from cellaggregates of the strain TU cell line. The thiamine requirementof plant cultured cells seems to be associated with the degreeof dedifferentiation of the cells rather than the kind of plant.In general, the higher the degree of redifferentiation of thecells, the higher is their thiamine level and the less theyrequire externally supplied thiamine. ¹Present address: Section of Phytochemical Research, Eisai Co.,Ltd., Kawashima, Gifu 483, Japan. (Received December 15, 1978; )     

Regulation of retinol-binding protein metabolism in cultured rat liver cell lines

Retinol-binding protein (RBP), the plasma transport protein for vitamin A, is synthesized and secreted by the liver. In vitamin A deficiency, RBP secretion is blocked, leading to low serum and high liver levels of RBP. Administration of retinol to the intact rat stimulates a rapid secretion of RBP from liver into serum. We explored the use of a liver cell culture system to study the regulation of the synthesis and secretion of RBP. We found two lines of differentiated rat hepatoma cells, MH₁C₁ and H₄ II EC₃ (H₄), that synthesized RBP during culture in vitro. The net synthesis of RBP was a function of the number of cells per dish and the duration of incubation. Both cell lines synthesized RBP when incubated in Neuman and Tytell's Serumless Medium (NTS medium), while the MH₁C₁ cells also synthesized RBP in Ham's F-12 medium with added serum. A relatively large proportion (14–56%) of the RBP was retained within the cells when they were incubated in the vitamin A-free NTS medium alone. Addition of serum to NTS medium stimulated the release of RBP from the cells into the medium and also increased the net synthesis of RBP. These effects were not due to the increased adhesion of the cells to the petri dish. Addition of retinol (at levels of 0.35 or 3.5 nmole/ml) to the NTS medium resulted in the stimulation of RBP secretion from the cells into the medium and an increase in the net synthesis of RBP. By contrast, retinol had no effect on either the net synthesis or the cell-to-medium distribution of rat serum albumin. The data from these cell lines in culture suggest that retinol has a specific regulatory effect on RBP metabolism. These cells thus resemble the normal rat liver cell in vivo in regard to the known regulation of RBP metabolism.     

Presence of human papillomavirus genome in human tumor cell lines and cultured cells.

A series of 47 human carcinoma cell lines and their cultured cells were examined for human papillomavirus (HPV) genomes with the use of an HPV detection kit (DNA-RNA hybridization, mixed HPV DNA probe of types 6, 11, 16, 18, 31, 33 and 35). Four of 8 cases of mild dysplasia, 3 of 9 cases of severe dysplasia, 3 of 7 cases of carcinoma in situ, 3 of 15 cases of uterine carcinoma and 5 of 6 cases of condyloma acuminatum were shown to contain the HPV DNA genome in primary cultured cells, while HPV was not detected in the third-passage cells except for the three cases of large cell, nonkeratinizing squamous cell carcinoma. HPV was also not detected in such normal tissues as uterine cervical squamous epithelium, uterine cervical columnar epithelium and endometrium. The presence of HPV DNA genomes was detected consistently in the passages of three lines (SKG-II, HKMUS and HKTUS; large cell nonkeratinizing squamous cell carcinomas of the uterine cervix) with the use of the Southern Blot method (DNA-DNA hybridization, mixed HPV probe of types 6, 11, 16 and 18). HPV type 16 DNA was detected in HKTUS, and HPV type 18 DNA was found in SKG-II and HKMUS. The other 44 cell lines, including ovarian carcinoma, endometrial carcinoma, sarcoma, gastric cancer, pancreatic cancer and rectal cancer, were negative for the HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, HPV-33 and HPV-35 genomes under stringent hybridization conditions.     

Peptidomic analyses of mouse astrocytic cell lines and rat primary cultured astrocytes

Astrocytes play an active role in the modulation of synaptic transmission by releasing cell-cell signaling molecules in response to various stimuli that evoke a Ca(2+) increase. We expand on recent studies of astrocyte intracellular and secreted proteins by examining the astrocyte peptidome in mouse astrocytic cell lines and rat primary cultured astrocytes, as well as those peptides secreted from mouse astrocytic cell lines in response to Ca(2+)-dependent stimulations. We identified 57 peptides derived from 24 proteins with LC-MS/MS and CE-MS/MS in the astrocytes. Among the secreted peptides, four peptides derived from elongation factor 1, macrophage migration inhibitory factor, peroxiredoxin-5, and galectin-1 were putatively identified by mass-matching to peptides confirmed to be found in astrocytes. Other peptides in the secretion study were mass-matched to those found in prior peptidomics analyses on mouse brain tissue. Complex peptide profiles were observed after stimulation, suggesting that astrocytes are actively involved in peptide secretion. Twenty-six peptides were observed in multiple stimulation experiments but not in controls and thus appear to be released in a Ca(2+)-dependent manner. These results can be used in future investigations to better understand stimulus-dependent mechanisms of astrocyte peptide secretion.     

The effect of caffeine on post-replication repair and survival in two L5178Y cell lines with different sensitivities to UV irradiation

2 Strains of murine lymphoma L5178Y cells that varied from the point of view of sensitivity to UV irradiation (mean lethal doses: 3.6 and 8.5 J/m2 for L5178Y-R and L5178Y-S cells, respectively) also differed with respect to sensitization by caffeine. L5178Y-S cells were sensitized to UV irradiation by 0.75 mM caffeine, whereas in the same conditions L5178Y-R cells were not sensitized. Sedimentation analysis of the newly synthesized DNA indicated UV-induced gap formation in L5178Y-S cells only. The subsequent gap filling was inhibited by caffeine. Exposure to UV irradiation induced no gaps in L5178Y-R cells. However, when caffeine was added immediately after irradiation, DNA with reduced molecular weight was found in irradiated cells of both strains after a 2-h chase. On the other hand, caffeine inhibited elongation of undamaged DNA strands in neither of the 2 cell strains.     

Effects of Hoechst 33342 on survival and growth of two tumor cell lines and on hematopoietically normal bone marrow cells

Our objective in this investigation was to determine whether Hoechst 33342, which is widely recognized as a DNA specific fluorochrome for living cells, is in fact nontoxic and kinetically nonperturbing at dye concentrations required to achieve acceptable DNA distributions. Three cell types were tested: HeLa S-3; SK-DHL2, a human lymphoma cell line; and hematopoietically normal human bone marrow cells. In the third system, only the cloning efficiencies were determined. Results differed considerably for the different cell types. While HeLa cells yielded excellent DNA distributions and were almost completely resistant to the cytotoxic and cytokinetic effects of the dye, SK-DHL2 cells were highly sensitive to the fluorochrome even at dye concentrations which produced very poor DNA distributions. Human bone marrow cells were intermediate in their stainability and toxicity response, and acceptable DNA distributions could be obtained at the nontoxic dye concentration of 2.5 muM. Clearly, different cell types differ considerably with respect to their cytotoxic and kinetic responses to Hoechst 33342. In some cases it may not be possible to ensure adequate staining of the cells for flow cytometry without significantly altering their viability and/or proliferative behavior.