Tore Lindmo

CELLULAR AND NUCLEAR VOLUME DURING THE CELL CYCLE OF NHIK 3025 CELLS

The distribution of cellular and nuclear volume in synchronous populations of NHIK 3025 cells, which derive from a cervix carcinoma, have been measured by electronic sizing during the first cell cycle after mitotic selection. Cells given an X‐ray dose of 580 rad in G1, were also studied. During the entire cell cycle the volume distribution of both cells and nuclei is an approximately Gaussian peak with a relative width at half maximum of about 30%. About half of this width is due to imperfect synchrony whereas the rest is associated with various time invariant factors. During S the mean volume of the cells grows exponentially whereas the nuclear volume increases faster than for exponential kinetics. Hence, although cellular and nuclear volumes are closely correlated, their ratio does not remain constant during the cell cycle. Volume growth during the first half of G1 is negligible especially for nuclei where the growth appears to be closely associated with DNA‐synthesis. For unirradiated cells the growth of cellular and nuclear volume is negligible also during G2+ M. In contrast, the X‐irradiated cells continue to grow during the 6 hr mitotic delay with a rate that is constant and about half of that observed in late S. Hence, radiation induced mitotic delay does not appear merely as a lengthening of an otherwise normal G2. During G1 and S the irradiated cells were identical to unirradiated ones with respect to all the parameters measured.

Cell cycle characteristics of synchronized and asynchronous populations of human cells and effect of cooling of selected mitotic cells.

The method of synchronizing cells by means of mitotic selection has been adapted to the human line NHIK 3025. Increase in cell number as a function of time in asynchronous and synchronous populations was studied as well as mitotic index as a function of time after selection of synchronized populations. Phase durations of the cell cycle of synchronous populations were determined by 3 H‐thymidine incorporation and scintillation counting. The relative phase durations of exponentially growing asynchronous populations were determined by mathematical analysis of DNA‐histograms recorded by flow cytofluorimetry. Both the generation time and the various phase durations of the cell cycle were found to be the same in asynchronous and synchronous populations. It was found that NHIK 3025 cells are damaged by cooling to 4 and 0°C so that cooling of selected cells in order to increase the yield would reduce the quality of the synchronized populations.

REGENERATIVE PROLIFERATION OF MOUSE EPIDERMAL CELLS FOLLOWING ADHESIVE TAPE STRIPPING

The proliferating cells of mouse epidermis (basal cells) can be separated from the non‐proliferating cells (differentiating cells) (Laerum, 1969) and brought into a mono‐disperse suspension. This makes it possible to determine the cell cycle distributions (e.g. the relative number of cells in the G^ S and (G2+ M) phases of the cell cycle) of the basal cell population by means of micro‐flow fluorometry. To study the regenerative cell proliferation in epidermis in more detail, changes in cell cycle distributions were observed by means of micro‐flow fluorometry during the first 48 hr following adhesive tape stripping. 3H‐TdR uptake (LI and grain count distribution) and mitotic rate (colcemid method) were also observed. An initial accumulation of G2 cells was observed 2 hr after stripping, followed by a subsequent decrease to less than half the control level. This was followed by an increase of cells entering mitosis from an initial depression to a first peak between 5 and 9 hr which could be satisfactorily explained by the changes in the G2 pool. After an initial depression of the S phase parameters, three peaks with intervals of about 12 hr followed. The cells in these peaks could be followed as cohorts through the G2 phase and mitosis, indicating a partial synchrony of cell cycle passage, with a shortening of the mean generation time of basal cells from 83‐3 hr to about 12 hr. The oscillations of the proportion of cells in G2 phase indicated a rapid passage through this cell cycle phase. The S phase duration was within the normal range but showed a moderate decrease and the Gj phase duration was decreased to a minimum. In rapidly proliferating epidermis there was a good correlation between change in the number of labelled cells and cells with S phase DNA content. This shows that micro‐flow fluorometry is a rapid method for the study of cell kinetics in a perturbed cell system in vivo.

Growth characteristics of human melanoma xenografts

Abstract. The growth of twelve human malignant melanomas in athymic nude mice was studied. Gompertz curves were fitted to volumetric growth data. DNA histograms were obtained with flow cytometry. Each of the twelve melanomas exhibited a characteristic growth pattern, indicating that inherent properties of the tumours are important for the growth control. The theoretical maximum volumes (Vmax) ranged from 208 to 12,900 mm3, the volume doubling times (Td) from 2.8 to 15.3 days (V= 50 mm3) and from 3.8 to 64.6 days (V= 200 mm3), and the fraction of cells in S from 5 to 21%. Tumours with short Td were characterized by a higher growth fraction and probably by a lower cell loss factor than those with long Td. The growth was also influenced by the nude mouse host, as indicated by the values for Vmax which were similar to those reported for mouse tumours (geometric mean = 8100 mm3), but considerably lower than the volumes of many tumours in man. Also the Td‐values for the xenografts were generally lower than those reported for tumours in man, presumably due to a lower cell loss factor. During serial transplantation the growth rate of one of the melanomas increased abruptly, probably because of both an increased growth fraction and a reduced cell loss factor. The latter result demonstrates the necessity of keeping basic biological parameters of xenografts under observation during serial transplantation.

DELAY OF CELL CYCLE PROGRESSION AFTER X-IRRADIATION OF SYNCHRONIZED POPULATIONS OF HUMAN CELLS (NHIK 3025) IN CULTURE

The effect of X‐irradiation on the cell cycle progression of synchronized populations of the human cell line NHIK 3025 has been studied in terms of the radiation‐induced delay of DNA replication and cell division. Results were obtained by flow cytometric measurement of histograms of cellular DNA content and parallel use of conventional methods for cell cycle analysis, such as pulse labelling with [3H]thymidine and counting of cell numbers. The two sets of methods were generally in good agreement, but the advantages of employing two independent techniques are pointed out.

Inhibition of cell-cycle progression by acute treatment with various degrees of hypoxia: Modifications induced by low concentrations of misonidazole present during hypoxia

The effect on cell-cycle progression in various phases of the cell cycle caused by an acute exposure to hypoxia in absence and presence of misonidazole (MISO) was investigated. Exponentially growing and synchronized cells of the human line NHIK 3025 were exposed to different degrees of hypoxia for a short period (1.5 or 3 h). The cell-cycle progression was studied both during and after hypoxia by flow-cytometric recording of DNA-histograms from treated and untreated cells. The rate of cell-cycle progression was reduced during hypoxia only if the O2-concentration was below 1000 ppm. The inhibition was phase specific with a strong effect in S (reduced DNA-synthesis), and a specific cell-cycle inhibition in late G1, probably at the G1/S-border. For cells inhibited (or arrested for extreme hypoxia) at the G1/S-border, the cell-cycle progression changed back to normal shortly after aerobic conditions were re-established. For cells rendered hypoxic and inhibited during S, hypoxia exerted a lasting effect expressed by a low cell-cycle progression rate even after aerobic conditions were re-established. This effect was strongly dependent on both the degree and the duration of the hypoxic treatment. The presence of a low concentration of MISO (0.05 mM) during hypoxia did not affect the cell-cycle progression during hypoxia at any O2-concentration. For cells rendered hypoxic during S, however, MISO (0.05 mM) counteracted the lasting effect of hypoxia for all concentrations of O2 where this lasting effect was observed.

Inactivation of human osteosarcoma cells in vitro by 211At-TP-3 monoclonal antibody: comparison with astatine-211-labeled bovine serum albumin, free astatine-211 and external-beam X rays.

The potential usefulness of alpha-particle radioimmunotherapy in the treatment of osteosarcoma was studied in vitro by using the monoclonal antibody TP-3 and cells of three human osteosarcoma cell lines (OHS, SAOS and KPDX) differing in antigen expression. Cell survival curves were established after treatment with (a) 211At-TP-3 of different specific activities, (b) 211At-labeled bovine serum albumin (BSA), (c) free 211At and (d) external-beam X rays. The three osteosarcoma cell lines showed similar survival curves, whether treated with external-beam X rays, 211At-BSA or free 211At. The D0s were lower for free 211At than for 211At-BSA. The survival curves for 211At-TP-3 treatment, on the other hand, differed significantly among the cell lines, suggesting that sensitivity to 211At-TP-3 treatment was governed by cellular properties other than sensitivity to external-beam X rays. The cellular property most important for sensitivity to 211At-TP-3 treatment was the antigen expression. Cell inactivation after 211At-TP-3 treatment increased substantially with increasing specific activity of the 211At-TP-3. At high specific activities, the cytotoxic effect of 211At-TP-3 was significantly higher than that of 211At-BSA. In conclusion, 211At-TP-3 has the potential to give clinically favorable therapeutic ratios in the treatment of osteosarcoma.

Cell Kinetic Characteristics In Different Parts of Multicellular Spheroids of Human Origin

The growth fraction, the cell cycle time, and the duration of the individual cell cycle phases were determined as a function of distance from the surface of multicellular spheroids of the human cell line NHIK 3025. the techniques employed were percentage of labelled mitoses and labelling index measurements after autoradiography and flow cytometric measurements of DNA histograms. to separate cell populations from the different parts of the spheroid, fractionated trypsinization was employed.

Effect of single dose irradiation on the proliferation kinetics in a human malignant melanoma in athymic nude mice.

The effect of single dose irradiation on the proliferation kinetics in a human malignant melanoma grown in the athymic mutant nude mouse was analysed. DNA-histograms were obtained with flow cytofluorometry. Percentage labelled mitoses curves were established by the use of conventional autoradiographic techniques. Changes in the fraction of clonogenic cells with time after irradiation were measured in vitro in soft agar. In non-irradiated tumours the fraction of cells in G1/G0, S and G2 + M was 66.21 and 13 per cent, respectively. The median duration of G1, S, G2, M and Te was 19.0 h, 13.3 h, 5.0 h, 1.0 h and 41.4 h, respectively. The growth fraction was calculated as 0.66 and the cell loss factor as 0.67. The growth fraction was increased after irradiation and the cell cycle time reduced, due to a shortening of G1. These effects were dose dependent and decreased with time after exposure, but were still present after the tumours had resumed a continuous volume growth. The rate of volume growth was slower for irradiated tumours than for non-irradiated tumours of the same size, due to a larger cell loss factor for the former.

Progress through G1 and S in relation to net protein accumulation in human NHIK 3025 cells

We have investigated whether human NHIK 3025 cells are dependent upon a net increase in cellular protein content in order to traverse G1 and S. The increase in DNA and protein content was studied by means of two-parameter flow cytometry using populations of cells synchronized by mitotic selection. By adding 1 microM cycloheximide to the medium protein synthesis was partially inhibited, resulting in negligible net accumulation of protein. The cells were able to enter S and progress through S under such conditions. The latter was the case whether the cells had been accumulating protein during G1 or not. The results further indicate that the larger cells enter S earlier and traverse S at a higher rate than the smaller cells. Our conclusion is that net accumulation of protein does not seem to be a prerequisite for traverse through G1 and S, i.e. DNA replication may be dissociated from the general growth of cell mass.