Tumour cell recruitment of the JB-1 and L 1210 ascites tumour determined directly by double labelling with [14C]- and [3H]-thymidine

Tumour cell recruitment of the JB-1 and L 1210 ascites tumour has been demonstrated directly by a double-labelling method with [14C]- and [3H]-thymidine (TdR). After [14C]-labelling of all proliferating tumour cells by multiple injections of [14C]TdR, recruitment of resting cells was stimulated by removal of the majority of tumour cells, i.e. by maximum aspiration of ascitic fluid. The number of recruited resting cells in the remaining tumour that re-enter the cell cycle after stimulation was demonstrated directly by a single injection of [3H]TdR given at different times after stimulation. The increase in the percentage of purely [3H]-labelled cells, i.e. recruited cells, with increasing time after stimulation, shows that recruitment is not a synchronous but a continuous process, the maximum of which occurs earlier in the case of the L 1210 than the JB-1 tumour. This suggests that there seems to be a relationship between the time required for maximum recruitment and the corresponding cell cycle parameters of the unperturbed tumour. There is a transitory increase of the growth fraction to about 100% and a considerable shortening of the cycle time at the maximum of recruitment.     

Tumour Cell Recruitment of the Jb-1 and L 1210 Ascites Tumour Determined Directly By Double Labelling With [14C]- and [3H]-Thymidine

Abstract. Tumour cell recruitment of the JB-1 and L 1210 ascites tumour has been demonstrated directly by a double-labelling method with [¹⁴C]- and [³H]-thymidine (TdR). After [¹⁴C]-labelling of all proliferating tumour cells by multiple injections of [¹⁴C]TdR, recruitment of resting cells was stimulated by removal of the majority of tumour cells, i.e. by maximum aspiration of ascitic fluid. the number of recruited resting cells in the remaining tumour that re-enter the cell cycle after stimulation was demonstrated directly by a single injection of [³H]TdR given at different times after stimulation. the increase in the percentage of purely [³H]-labelled cells, i.e. recruited cells, with increasing time after stimulation, shows that recruitment is not a synchronous but a continuous process, the maximum of which occurs earlier in the case of the L 1210 than the JB-1 tumour. This suggests that there seems to be a relationship between the time required for maximum recruitment and the corresponding cell cycle parameters of the unperturbed tumour. There is a transitory increase of the growth fraction to about 100% and a considerable shortening of the cycle time at the maximum of recruitment.     

CYTOKINETIC ANALYSIS OF THE JB-1 ASCITES TUMOUR AT DIFFERENT STAGES OF GROWTH

The cell kinetics of the murine JB-1 ascites tumour have been investigated on days 4, 7 and 10 after transplantation of 2·5 × 10⁶ cells. The experimental data, growth curve, percentage of labelled mitoses curves, continuous labelling curves and cytophotometric determination of single-cell DNA content have been analysed by means of a mathematical model for the cell kinetics. The important result was the existence of 8% non-cycling cells with G₂ DNA content in the 10-day tumour, while only 0·2 and 0% were observed in the 7- and 4-day tumours, respectively. The doubling times determined from the growth curve were 22·8, 70 and 240 hr, respectively, in the 4-, 7- and 10-day tumours. Growth fractions of 76, 67 and 44% were calculated for the same tumour ages. The mean cell cycle time increased from 14 to 44 hr from day 4 to 7 due to a proportional increase in the mean transit time of all phases in the cell cycle. In the 10-day tumour, the mean cell cycle changed to 41 hr and T _G1 decreased to 0·5 hr. The cell production rate was 4·3%/hr in the 4-day tumour, 1·2%/hr in the 7-day tumour and 1·0%/hr in the 10-day tumour. The cell loss rates in the same tumours were 1·3, 0·2 and 0·7%/hr, respectively. The analysis made it probable that the mode of cell loss was an age-specific elimination of non-cycling cells with postmitotic DNA content.     

Employment of synchronized cells and flow microfluorometry in investigations on the JB-1 ascites tumour chalones.

In most experimental ascites tumours the growth rate decreases with increasing age and cell number. This decrease is caused by a prolongation of the cell cycle and an increasing accumulation of non-cycling cells in resting (or quiescent) G1 and G2 compartments. In cell-free ascitic fluid from the JB-1 ascites tumour in the plateau phase of growth lowmolecular-weight substances have been found which reversibly and specifically arrest JB-1 cells in G1 and G2. The present paper describes an in-vitro model for testing the effect of the humoral growth inhibitors contained in the ascitic fluid. The test system is based on synchronized JB-1 cells analysed by flow-through cytofluorometry. Addition to the synchronous cells of a ultrafiltrate (less than 50000 Daltons) of the JB-1 ascitic fluid was found to induce a complete, but temporary arrest of the cells at the G1-S border.     

PROLIFERATION KINETICS OF AN EXPERIMENTAL ASCITES TUMOUR OF THE MOUSE

The cell cycles of an experimental ascitic tumour of the C3H mouse (NCTC 2472) were determined at various times after the intraperitoneal injection of 10⁶ cells. It was found that, contrary to results in solid NCTC 2472 tumours, obtained with the same NCTC cells, the duration of the cell cycle and its phases lengthened with the age of the tumour while the growth fraction remained relatively constant. G₁ was the first phase to lengthen, while later T_s and T_G2 increased also. The amount of DNA per cell was determined by cytospectrophotometry. This method provides data on the evolution during growth of the relative number of cells in each phase of the cell cycle.     

Resistance of ascitic fluid immunosuppressive factor DNA to pancreatic deoxyribonuclease]

It was found that incubation of Ehrlich tumour ascitic fluid with pancreatic DNAse does not result in a destruction of 5S DNA in the immunodepressive factor. After phenol deproteination of the ascitic fluid the resistance of this nucleic factor to DNAse is lost. It is possible that the native nucleic factor is represented by DNP and contains a protein component. During polyacrylamide gel electrophoresis the mobility of the nucleic factor labelled with [3H]-thymidine and isolated after chromatography on hydroxyapatite is found to be nearly the same as that for transferine.     

Cell kinetic studies on the JB-1 ascites tumour of the mouse

Abstract. The FLM method, modified by double labelling with [³H]- and [¹⁴C]-thymidine, has been applied to the 4-day old JB-1 ascites tumour of the mouse. It results in well separated waves of purely [³H]- and purely [¹⁴C]-labelled mitoses, which show a remarkable asymmetry with long tails to the right. The following values for the mean transit times of the cells have been derived from this FLM curve, for a tumour age of 4–6 days: T_C= 32.5 hr, T_S= 16.7 hr, T_G1= 3.7 hr, T_G1= 11.0 hr and T_M= 1.1 hr. A further evaluation of the FLM curve, however, is difficult, due to the non-stationary growth of the tumour. A number of other experimental findings (growth curve, decrease of the labelling and mitotic index with increasing tumour age, two single-labelled FLM curves starting 4 and 6 days after tumour inoculation) indicate that the cell cycle time increases during the experimental period of the double-labelled FLM curve (about 2 days). A lengthening of the cycle time should result in an increasing enlargement of the areas under the waves of the modified FLM curve. However, such an increase in area has not been found; the areas are constant. All the results of the present cell kinetic studies would be consistent if it were postulated that the cell cycle time lengthens with increasing tumour age up to about 4 days after inoculation, then remains relatively constant at between 4 and 6 days and thereafter increases again. Short-term double labelling experiments suggest that this is actually the case. Under the assumption of nearly constant phase durations during the 5th and 6th day of tumour growth further conclusions can be drawn from the modified FLM curve. In particular, it follows that the transit times of the cells through successive cycle phases are uncorrelated and the variances of the transit times through a cycle phase are proportional to the duration of this phase.     

CYTOKINETICS OF TUMOUR AND ENDOTHELIAL CELLS AND VASCULARIZATION OF LUNG METASTASES IN C3H/HE MICE

A model of lung metastases was developed using intravenous injection of tumour cell aggregates of spontaneous C3H/He mammary tumours in syngeneic mice. the growth rate of lung tumours decreased with increasing tumour volume, with mean host survival of 46 days. the cytokinetics of individual tumours ranging between 0.004 and 4.2 mm³ in volume were studied. the labelling index (LI) ranged between 12 and 17%, the DNA synthesis time (T_s) being 9–10 hr. the growth fraction (GF) ranged between 26 and 38%. the cell cycle time (T_c) was found to be 18–19 hr. the LI and the GF decreased with increasing tumour volume doubling time (T_d). No correlation was found between the tumour volume and T_c. the LI of endothelial cells within these tumours, ranging between 0.004 and 4.2 mm³ in volume was 14–15% and endothelial cell proliferation was not affected by tumour growth. Vascular parameters were also determined for these tumours as a function of tumour volume. Vascular volume increased with increasing tumour size while the percentage of capillary vessels decreased. the cellular volume to capillary volume ratio increased with increasing tumour volume. Necrosis was observed in 0.27 mm³ tumours and increased with increasing tumour size. The results from these studies suggested that the age-dependent decrease in proliferative activity of tumour cells growing in the lung is related to change in effective vascularity.     

Studies on Ehrlich ascites tumour cells: DNA synthesis,and template activity of chromatin for DNA polymerase

Summary Chromatin obtained from Ehrlich ascites cells on different days after cell inoculation has been assayed for its template activity with added DNA polymerase 1. We have found that the template activity is 2 times higher in 7–8 day cell chromatin than in 4-day chromatin. Studies with added polylysine indicate that this increase reflects an increase in initiation sites rather than in accessibility to the enzyme. We have measured the growth fraction, mitotic index and rate of DNA chain growth in the intact cells. The results show that there is a large decrease in growth fraction with age of tumour, the number of cells dropping out of cycle approximately doubling over the period studied. The overall rate of chain growth decreases in the later stages of growth but in a small proportion of cells there is an increase in rate with fewer replicons involved in DNA synthesis. We suggest that in the ascites cells there is a decrease in level of repair and replicative enzymes with age of tumour; this would account both for the increase in initiation sites in the chromatin DNA, for the decrease in number of cells in cycle and for the overall decreased rate of chain growth.     

Growth stimulation of rat fetal hepatocytes in response to hepatocyte growth factor: modulation of c-myc and c-fos expression.

Hepatocyte growth factor, which is a potent growth factor for primary cultured adult hepatocytes, strongly stimulated DNA synthesis of rat fetal (20-day of gestation) hepatocytes. Its mitogenic capacity, measured as (3H)-thymidine incorporation into acid precipitable material was dose dependent, being detectable at 1 ng/ml and maximal at 5 ng/ml. Over 15% of the cells entered into S-phase and mitosis as judged by flow cytometric analysis of the cell cycle. HGF had additive effects with transforming growth factor-alpha, whereas transforming growth factor-beta strongly inhibited DNA synthesis of fetal hepatocytes stimulated by HGF. HGF induced c-fos and c-myc expression in a time-dependent manner, with a maximum at 30 min for c-fos and 8 h for c-myc. These results suggest that HGF may act as a proliferative factor during fetal liver growth.     

Differential effects of nitrogen mustard in vivo on the cancer and host cells in MCa-11 tumours

We analysed the effects of nitrogen mustard (HN₂) on the growth, cell cycle distributions, and ratios of tumour cells to host cells for MCa-11 tumours grown in vivo. Treatment of tumour-bearing BALB/c mice with 3 mg/kg of HN₂ produced a significant slowing of MCa-11 tumour growth. Seventy-two hours after treatment in vivo with either 3 or 4 mg/kg of HN₂, the host cells in the treated tumours showed a significantly decreased G₀/G₁ peak and an increased G₂/M peak (P < 0.01), whereas the cancer cells in the treated tumours showed significant increases in the G₀/G₁ peak coupled with relatively decreased proportions of S and G₂/M tumour cells (P < 0.001). The ratio of the total number of cancer cells to the total number of host cells in the tumours was significantly increased 72 h after HN₂ administration (P<0.01). Thirty-two days after treatment with HN₂, the cell cycle distributions of the host and tumour cells in the treatment and control tumours had returned to being identical, but the ratio of the total number of cancer cells to the total number of host cells remained increased in the treated tumours (P<0.01). These results show that the administration in vivo of HN₂ can lead to entirely different cell cycle effects for the host and cancer cells in the same tumour, and that the partial growth arrest of MCa-11 tumours from HN₂ treatment may be due in part to the preferential destruction of host cells rather than solely to a direct cytotoxic effect on the cancer cells.     

Cell kinetics of a rat thyroid transplantable tumour

Changes in morphology and cell kinetics are described in a rat thyroid transplantable tumour (TTT) during the first few transplant generations. The growth of TTT in animals was possible only with an increased circulation level of the thyroid stimulating hormone (TSH). With serial transplantation subcutaneously in isologous animals, the morphology of TTT changed dramatically from that of a follicular tumour in the 3rd passage to become, by the 9th generation, a poorly differentiated tumour with a trabecular arrangement of cells. This change in tumour morphology was accompanied by an increase in the number of proliferating cells--mitotic index (MI), [3H]thymidine labelling index (LI), growth fraction (GF)--and cell loss factor (O) as well as a decrease in the cell cycle time (Tc) and potential population doubling time (TPD). TTT belongs to the class of tumours with a low proliferative activity and might be used in a variety of cell kinetic, radiobiological and chemotherapy studies.     

Proliferative changes in two murine tumours in response to single or fractionated doses of X-rays

Abstract. Studies were carried out to investigate proliferative changes in two murine experimental tumours in response to radiation. Results were generated using bro-modeoxyuridine labelling and flow cytometry. This study demonstrates the possible ambiguity of previous studies using tritiated thymidine in which inability to discriminate normal and tumour cell components in murine tumours may lead to different values for cell kinetic parameters. In particular, the sarcoma F appeared to have a growth fraction of 0.62 when all cells were considered; in reality the growth fraction of the tumour cells only (based on DNA content discrimination) was close to unity. Radiation, administered either as single or fractionated doses, caused little change in the proliferative characteristics of the sarcoma F tumour but had profound effects on the adenocarcinoma Rhodesia tumour. Major changes were the accumulation of cells in G₂ for several days after the end of the radiation treatment in both tumours and a dramatic drop in labelling index of the Rhodesia tumour. In neither tumour was there any evidence to suggest an increase in tumour cell proliferation during or after the irradiations. The diploid cells within the sarcoma F tumour showed an initial depression of labelling index followed by a rapid increase overshooting the control labelling index at higher radiation doses. Much of the effects could be attributed to cell cycle delays.     

Proliferation kinetics of endothelial and tumour cells in three mouse mammary carcinomas

Abstract. An autoradiographic study of three corded mouse tumours is reported. The proliferation characteristics of both tumour cells and endothelial cells were studied. The doubling time of these three tumours differed by a factor of 2.6 but there was only a small difference in the intermitotic time. All three tumours showed a very high cell loss factor (˜0.80) and the differences in growth rate resulted mainly from differences in the growth fraction .
The endothelial cell proliferation rates differed markedly in the three tumours, with labelling indices ranging from 18% in the faster tumours to 4.5% in the slowest. The potential doubling times for endothelium, calculated from these values, were much slower than the tumour cell cycle time or the tumour potential doubling time, but were two to four times faster than the volume doubling time of the tumour.
It appears likely that the endothelial proliferation rate influences the growth fraction, but similar high cell loss factors can occur in tumours with a four-fold difference in endothelial cell production rates. Inadequate branching of blood vessels seems likely to be at least as important as inadequate production of endothelial cells. It is not possible to determine whether slow tumour cell production evokes a slower endothelial growth or vice versa.     

Ascitic and solid Ehrlich tumor inhibition by Chenopodium ambrosioides L. treatment

The leaves of Chenopodium ambrosioides L. [Chenopodiaceae] ('mastruz') have been indicated for the treatment of several diseases, among which the cancer. There are no results focusing the effect of C. ambrosioides treatment on tumor development in vivo. The aim of this study was to investigate the effect of treatment with C. ambrosioides on Ehrlich tumor development. Swiss mice were treated by intraperitoneal route (i.p.) with hydroalcoholic extract from leaves of C. ambrosioides (5 mg/kg) or with PBS (control group) 48 h before or 48 h later the Ehrlich tumor implantation. The tumor cells were implanted on the left footpad (solid tumor) or in the peritoneal cavity (ascitic tumor). To determine the solid tumor growth, footpad was measured each 2 days until the fourteenth day, when the feet were weighed. Ascitic tumor development was evaluated after 8 days of tumor implantation by quantification of the ascitic fluid volume and tumor cell number. The i.p. administration of C. ambrosioides extract before or after the tumor implantation significantly inhibited the solid and ascitic Ehrlich tumor forms. This inhibition was observed in ascitic tumor cell number, in the ascitic volume, in the tumor-bearing foot size and foot weight when compared to control mice. The treatments also increased the survival of tumor-bearing mice. In conclusion, C. ambrosioides has a potent anti-tumoral effect which was evident with a small dose and even when the treatment was given two days after the tumor implantation. This effect is probably related with anti-oxidant properties of C. ambrosioides.     

The relationship between the doubling time of human lung tumor volume and the labelling index]

The value of the labelling index (incubation with H3-thymidine) was compared with the doubling time of tumour volume (by repeated roentgenograms) for 3 tumours of human lungs: two squamous cell carcinomas taken at various differentiation levels and a hamartoma. The minimum doubling time (178 days) was shown for the squamous cell carcinoma with a lower differentiation level and the greatest labelling index value (19.0%). Hamartoma (a non-malignant tumour of lung) displayed the longest doubling time (1250 days) and the least labelling index value (3.8%).     

Cell Kinetic Studies of Chick Brain Cells In Culture: an Autoradiographic Study With [3H]- and [14C]-Thymidine

Labelling index, S-phase duration and cell-cycle time of proliferating brain cells from 6-day-old chick embryos in culture were investigated autoradiographically after labelling with [³H]- and/or [¹⁴C]-thymidine. the dissociated cells were cultured in the absence or in the presence of brain extract from 8-day-old chick embryos. Cultures contained essentially two cell types, which could be easily distinguished by the size of their nuclei: small nuclei identified as belonging to precursor cells of neurons and large nuclei corresponding to astroglial cells. the labelling index of astroglial cells (16.4%) was about 2 times higher than that of the neuronal cells (9.9%). Under the influence of brain extract the labelling index of neuroblasts was nearly doubled while that of the astroglial cells remained nearly unchanged. From double-labelling experiments with [³H]- and [¹⁴C]-thymidine, the same S-phase duration of about 7 hr was found for both cell types cultured with or without brain extract. A cell-cycle duration of 39 hr for neuronal and of 29 hr for astroglial cells was found. the cycle times remained constant under the influence of brain extract. From the measured data mentioned above, a growth fraction of 50% (neuroblasts) and 68% (astroglial cells) was calculated in control cultures without brain extract. After addition of brain extract, the growth fraction increased for both cell types (neuroblasts: 92%; astroglial cells: 80%). the results demonstrate that more cells proliferate in the presence of brain extract, but the durations of the S-phase and the cell cycle remain unchanged.     

The Effect of X-Irradiation On Cell Loss In Five Solid Murine Tumours, As Determined By the 125Iudr Method

The rate of cell loss in irradiated RIF-1, EMT6, KHJJ, B16 and KHT tumours was studied using the ¹²⁵IUdR loss technique. Administration of ¹²⁵IUdR preceded localized tumour irradiation by 2 days. Loss of tumour radioactivity was measured for 6–8 days after irradiation. the blood flow to some tumours was occluded during, and for 30 min following, injection of the label to measure the amount of radioactivity entering the tumour as a result of reutilization of label from the gut epithelia and influx of labelled host cells. Irradiation did not significantly alter the amount of radioactivity entering these clamped tumours during the 8–10 days after injection of ¹²⁵IUdR. This permitted comparison of irradiated and control groups based on the loss of radioactivity from the non-occluded tumours. Irradiation of RIF-1, EMT6, KHJJ or B16 tumours with doses of 600, 1400, 2400 or 4400 rads produced no significant increase in the rate of loss of tumour radioactivity. This suggested that, in the population of labelled cells, cell lysis following irradiation proceeded slowly. In contrast, KHT tumours showed a significant increase in loss rate following each radiation dose, although the increase was dose-independent. In all tumour systems, the constant rate of cell loss after radiation appeared to coincide with published reports of tumour growth responses after irradiation. the present data suggest that the manner of expression of radiation-induced cell killing results from the cellular proliferative status, i.e. whether a cell is cycling or non-cycling.     

Effect of monoclonal antibodies to early pregnancy factor (EPF) on the in vivo growth of transplantable murine tumours

Summary Neutralisation studies with monoclonal antibodies (mAbs) specific for early pregnancy factor (EPF) have shown it to be essential for the continuation of pregnancy in mice and the growth of some tumour cells in vitro. These studies report that the mAbs are also able to limit the growth of two murine tumour lines transplanted s. c. The development of MCA-2 tumours in CBA mice was unaffected by the injection of 1 mg anti-EPF IgM at the time of tumour cell inoculation. However, four doses of 500 µg anti-EPF, injected one dose per day for 4 days after tumour cell inoculation, significantly retarded tumour development such that no tumours were palpable on day 13. A similar dose regimen of control IgM had no effect on tumour size. Dose/response studies revealed that lower doses of anti-EPF administered after tumour cell inoculation were effective in retarding the growth of the MCA-2 tumours. The effect of anti-EPF mAb administration on the growth rate of palpable B16 tumours established s. c. in C57BL/6 mice was also determined. Tumours injected with 6 mg anti-EPF 5/341 or anti-EPF 5/333 mAbs showed significant decrease in the uptake of [³H]thymidine into tumour tissue, measured 16 h after injection. Furthermore, titration of sera for active EPF showed that a significant reduction in the EPF titre was associated with a significant inhibition of tumour DNA synthesis. Thus it appears that neutralisation of EPF retards tumour growth both in vitro and in vivo. In vitro the effects must be due to anti-EPF mAb interfering with a direct mechanism that contributes to the maintenance of cells in the active growing phase. However, in vivo host immunological mechanism that are modified to allow tumour survival may also be affected. The presence of EPF-induced suppressor factors curculating in the serum of tumour-bearing mice has been confirmed and the contribution of such factors to tumour progression must now be investigated.     

The Growth of the Emt6 Tumour In the Lungs of Balb C Mice Following Intravenous Inoculation of Tumour Cells From Culture

The growth of the EMT6 tumour in the lungs of Balb C mice has been studied following intravenous inoculation of different numbers of tumour cells taken from culture. At various times after injection of cells into mice, cell suspensions have been prepared from pairs of lungs and the number of in vitro colony forming cells assayed by plating into petri dishes. Following intravenous injection of 10⁵ cells, the time required for doubling of the number of clonogenic tumour cells appearing in the cell suspension is around 17 hr until such time that the total tumour cell population per set of lungs reaches 10⁸ cells (at 10–12 days). This doubling time has to be corrected for changes in ability to extract cells from the lungs into the cell suspension at various times and also for possible changes in plating efficiency in vitro. When these correction factors are applied, the most likely value for the doubling time of clonogenic tumour cells in the lungs is in the range 20–24 hr. This is a similar figure to that previously deduced for the EMT6 flank tumour during its microscopic period of growth. After reaching a total size of 10⁸ tumour cells, the time for doubling of the number of clonogenic tumour cells in the lung increases. During the later stages of tumour growth a good correlation is seen between total lung tumour weight and the number of clonogenic cells present. For the final 3–4 days of the initial period of rapid tumour growth, it is possible to carry out a haemocytometer count of tumour cells in the lung suspension and hence surviving fraction experiments may be carried out after various forms of treatment. In this way the response to treatment of microscopic tumour foci may be determined.