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Biomedical subjects

E Necas

Publications and source records attributed to E Necas.

At least 55 records · Page 3Linked to original sources
PubMed1989

Thymidine suicide and hydroxyurea kill ratios accurately reflect the proliferative status of stem cells (CFU-S).

We have previously found that on average 30% of hematopoietic stem cells (CFU-S) are in the S phase, which is at least three times the value published by others. Therefore, it seemed desirable to investigate the reliability of the methods used to measure the percentage of CFU-S in S phase. Various modifications of the [3H]thymidine suicide were tested and it could be demonstrated that results were not affected by them. Furthermore, results obtained with the [3H]thymidine suicide were compared to those obtained with methods utilizing hydroxyurea to kill CFU-S in S phase. The [3H]thymidine- and hydroxyurea-based methods gave parallel results. The parallel behavior validated all of these methods as reliable indicators of the turnover of CFU-S and presumably other stem cell populations.

Animals↗
PubMed1988

The spleen colony technique. I. Correction for the overlap effect and sources of error in CFU-s determination.

A linear model for the errors of the 'spleen colony' assay for haemopoietic stem cells has been derived. The components emerging from the model are interpreted and practical recommendations given for interpreting measurements made with this assay. The model permits correction for the effect of overlapping colonies and gives average errors for single measurements of the number of CFU-s. More reliable and more precise information can be obtained using this model. The spleen colony technique detects a population of immature precursor cells designated as CFU-s (Till & McCulloch, 1961). The relative error of measurement is often large when compared with the changes in the phenomena studied. Consequently a better knowledge of the errors of this technique is highly desirable. This paper should be regarded as an extension of the previous analysis of Till (1972). The theory for the errors of the spleen colony technique was applied to 905 determinations of the CFU-s numbers performed on random-bred mice. Data from random-bred mice rather than those from inbred mice have been used because the error components can be expected to be larger and, consequently, more easily detectable. The model of errors has also been validated using data published by Till (1972) and has subsequently been applied to data from several inbred mice strains (Znojil & Necas, 1988).

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PubMed1988

Non-circadian rhythm in proliferation of haematopoietic stem cells.

The proportion of haematopoietic stem cells (CFU-s) engaged in DNA synthesis was determined by means of the [3H]-thymidine [( 3H]TdR) suicide technique during recovery of bone marrow from the damage caused by a sublethal total body irradiation. In contrast with previous reports the [3H]TdR suicide rate was not permanently increased. It was observed that CFU-s passed through S phase in synchronous waves, following a dose of irradiation of 1.5 Gy. After a dose of 2.6 Gy, there was only one initial wave of increased CFU-s sensitivity to the action of [3H]TdR. Following the depression occurring 26 hr after the irradiation with 2.6 Gy, the proportion of CFU-s killed by the [3H]TdR was permanently increased until 5-6 days after irradiation. Thereafter large differences in the [3H]TdR suicide data were observed among individual mice. Evidence was obtained that individual mice, which had been irradiated by a dose of 2.6 Gy 8-9 days before, had identical values of the CFU-s [3H]TdR suicide rate in the bone marrow from different bones of the lower extremities. The recurrence of the synchronous waves in CFU-s passage through the cell cycle was recorded when the CFU-s population regenerated to only about 10% of its normal value. These waves were obviously not related to a particular time of the day and, consequently, they did not represent the circadian rhythm. It is concluded that the synchronous waves in which CFU-s proliferation occurred reflected the action of the control mechanism on CFU-s proliferation. This mechanism should be endowed with an important systemic component besides locally operating factors.

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PubMed1988

Simulation of CFU-s kinetics after irradiation.

A simulation model of the CFU-s population is used to interpret data from experimental studies of bone marrow recovery after irradiation. The model includes an original hypothesis that the proliferation rate in the CFU-s population depends on the number of DNA-synthesizing CFU-s. It is assumed that the DNA-synthesizing CFU-s produce a factor in the presence of which CFU-s enter the resting state G0 after mitosis and remain there for prolonged periods of time. The model can adequately reproduce complex CFU-s kinetics observed after severe damage caused by irradiation with a unique set of parameters.

Animals↗
PubMed1988

The spleen colony technique. III. Comparison of the overlap effect and of errors in CFU-s determination and the [3H]-thymidine suicide data for several strains of mice.

A mathematical model of errors of the spleen colony technique is applied to data obtained from four mouse strains and F1 hybrids. The variance of the colony counts was close to the Poisson distribution in inbred mice and F1 hybrids. However, it should be checked regularly. The magnitude of the error in CFU-s determination and of the estimations of the S phase fraction was derived, and is presented relative to the mean colony counts for all mouse strains studied. The optimum spleen colony counts are generally higher than those which are commonly used. However, the utilization of the optimum spleen colony counts requires a correction for the effect of colony overlap.

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PubMed1988

Bone marrow response to single small doses of irradiation: implications for stem cell functional organization.

The data collected in 107 experiments over 23 months have been pooled to indicate the responses of cellularity, stem cell (CFU-S) number, and CFU-S proliferation rate (CFU-S in S-phase) during early recovery of the bone marrow from 1.5 and 2.6 Gy irradiation. The bone marrow differentials and numbers of granulocyte-macrophage progenitors (CFC-GM) were determined after irradiation with 1.5 Gy. The CFU-S proliferation rate was examined also in mice irradiated with 3.5-5.0 Gy. The data are discussed in the light of various hypotheses about the functional organization of the CFU-S population. It is proposed that the control of the CFU-S production rate is primary rather than the control of CFU-S numbers.

Animals↗
PubMed1988

Stem cell number versus the fraction synthesizing DNA.

It has been widely held that the fraction of spleen colony-forming units (CFU-S) in DNA synthesis is inversely correlated with CFU-S numbers. In 750 measurements the expected negative slope of the linear regression of CFU-S in S-phase on CFU-S was found to be significant only when the measurements from irradiated mice were pooled. In contrast, a significant positive correlation was observed when the measurements from normal controls were pooled. A hypothesis is offered to account for the variable relationship between CFU-S and CFU-S in S. The hypothesis fully recognizes the crucial role of both parameters in normal and regenerative hemopoiesis.

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PubMed1987

CFU-S content and cycling rate in several strains of mice.

The number of CFU-S and the fraction synthesizing DNA have been measured in individual normal mice of several inbred strains. The data gathered during a period of 5 years have been subjected to analysis of variance. Large differences are shown to exist in the number of CFU-S in the femoral bone marrow of individual mice measured on the same day. These differences are greater if measurements are performed on different days. The fraction of DNA synthesizing CFU-S was on average 30% in these normal mice. The range of measurements on both the same and different days was 0%-60%. The large differences in the fraction of CFU-S in S-phase were found even among mice coming from the same cage. This differs from many previous reports where 10% is given as the upper limit for the fraction of CFU-S synthesizing DNA in normal mice. It is suggested that the observed range of individual values from 0%-60% might reflect bursts of proliferation of CFU-S. Irradiation or hydroxyurea administration resulted in disappearance of low and medium values. All mice then had 50%-60% of CFU-S in S-phase.

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PubMed1985

The effect of zinc on mouse melanoma growth in vitro and in vivo.

The effect of Zn2+ on mouse melanoma growth in vitro and in vivo was studied. Under in vitro conditions the proliferation of a Cloudman mouse melanoma cell line was inhibited by zinc ions at 10(-4) M, as measured by 3H-thymidine incorporation and optical density of NaOH cell digests. However, in vivo it was not possible to suppress both B16 and Cloudman S91 melanoma growth in mice by the administration of zinc ions. There were no significant differences in tumor growth after subcutaneous inoculation between mice constantly receiving 0.1% zinc acetate or 0.05% zinc sulphate in their drinking water and control groups, nor was it possible to decrease the number of lung metastases by zinc treatment after intravenous inoculation of tumor cells. The increased dietary supply of Zn failed to influence the survival time of mice in both melanoma types studied. Preincubation in vitro of cell suspensions in 10(-3) M zinc acetate prior to injection inhibited melanoma development in vivo. This implies that the in vivo zinc levels did not reach the necessary cytotoxic concentration.

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PubMed1982

Analysis of the effect of hydroxyurea on stem cell (CFU-s) kinetics.

Hydroxyurea induces profound changes in the pluripotential haemopoietic stem cell (CFU-s) kinetics. The main feature of these changes is a synchronous entry of resting G0 CFU-s into the cell cycle. The analysis of the passage of the CFU-s cohort through the cell cycle has been largely based on the examination of the fraction of CFU-s which synthesize DNA in the S phase of the cell cycle. This analysis has, however, been hampered by the fact that both the sensitivity of the S phase CFU-s to hydroxyurea and their sensitivity in the [3H]thymidine suicide technique vary as the cells pass through the S phase. Methods which overcome these difficulties have been used in the experiments presented in this paper. It was demonstrated that hydroxyurea kills only about 80% of the S phase CFU-s. The sensitivity to hydroxyurea gradually decreases as the cells approach the middle part of the S phase and increases again as the cells enter the late portions of the S phase. The degree of CFU-s synchrony at the point of entry into and exit from, the S phase has been established. Mathematical analysis of the available data suggests that CFU-s pass through the S phase with a mean transit time of 4.79 hr (standard deviation, 1.45 hr). Hydroxyurea, administered in vivo, blocks CFU-s in the late G1 phase. The duration of this G1-S block, induced by a dose of 1000 mg of hydroxyurea per kg body weight, is approximately 2 hr. The CFU-s in the middle of the S phase, which survive hydroxyurea administration, are also blocked in their passage through the S phase. These cells, however, seem to finish the S phase with a delay of approximately 2 hr.

Animals↗
PubMed1982

Stem cell (CFUs) proliferation inhibitor in blood of mice injected with hydroxyurea.

Analysis of the mouse haemopoietic stem cell (CFUs) kinetics after hydroxyurea administration has provided an in vivo assay suitable for detection of factors which inhibit recruitment of non-proliferating G0-CFUs into cell cycle, or transit of CFU's through the G1 phase. Using this assay, it has been demonstrated that plasma obtained from mice which had received hydroxyurea approximately 12-14 hr previously, possesses a factor which inhibited the triggering of CFUs into the cell cycle. The appearance of this CFUs proliferation inhibitor occurred at a time when 60-70% of the CFUs were synchronized in the S phase of the cell cycle, as a consequence of hydroxyurea action. Some basic properties of the inhibitor were investigated.

Animals↗
PubMed1982

Stem cell (CFU-s) proliferation in sublethally irradiated mice.

The proliferation rate of haemopoietic stem cells (CFU-s) was followed after sublethal total body irradiation with 1 X 5 Gy. The [3H]-thymidine suicide technique was used to measure the CFU-s proliferation rate. The measurements extended from 10 min after irradiation up to 21 days. The CFU-s did not enter the DNA synthesis period (S-phase) shortly after irradiation, as had been previously suggested, but did so only with a delay of 14-16 hr. A large scatter of results was explained by an oscillatory pattern in CFU-s proliferation. The CFU-s prepared for cell division in synchronized waves, with a period of 20-22 hr.

Animals↗
PubMed1981

Decreased sensitivity to hydroxyurea and to [3H]thymidine suicide in the middle of the S phase.

Pluripotent haemopoietic stem cells (CFUs) move synchronously through the cell cycle in hydroxyurea-treated mice in a cohort 1--2 hr broad. Ten to fifteen hours after hydroxyurea they pass through S phase. DNA synthesis appears to be depressed 5--10 times when the cells are in the middle part of the S phase but does not seem to be completely interrupted. High concentrations of [3H]thymidine must be used for 'suicide' in order to achieve lethality for the cells with depressed DNA synthesis. At the time when DNA synthesis is depressed, the sensitivity of the cells to hydroxyurea also decreases. This may lead to a significant underestimation of the S phase fraction by the hydroxyurea method, because CFUs with low DNA synthesis rate are resistant to hydroxyurea although being in S phase.

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