Transit times through the cycle phases of jejunal crypt cells of the mouse

Mean transit times as well as variances of the transit times through the individual phases of the cell cycle have been determined for the crypt epithelial cells of the jejunum of the mouse. To achieve this the fraction of labelled mitoses (FLM) technique has been modified by double labelling with [3H] and [14C]thymidine. Mice were given a first injection of [3H]thymidine, and 2 hr later a second injection of [14C]thymidine. This produces a narrow subpopulation of purely 3H-labelled cells at the beginning of G2-phase and a corresponding subpopulation of purely 14C-labelled cells at the beginning of the S-phase. When these two subpopulations progress through the cell cycle, one obtains FLM waves of purely 3H- and purely 14C-labelled mitoses. These waves have considerably better resolution than the conventional FLM-curves. From the temporal positions of the observed maxima the mean transit times of the cells through the individual phases of the cycle can be determined. Moreover one obtains from the width of the individual waves the variances of the transit times through the individual phases. It has been found, that the variances of the transit times through successive phases are additive. This indicates that the transit times of cells through successive phases are independently distributed. This statistical independence is an implicit assumption in most of the models applied to the analysis of FLM curves, however there had previously been no experimental support of this assumption. A further result is, that the variance of the transit time through any phase of the cycle is proportional to the mean transit time. This implies that the progress of the crypt epithelial cells is subject to an equal degree of randomness in the various phases of the cycle

STABILITY OF DNA IN PURKINJE CELL NUCLEI OF THE MOUSE : An Autoradiographic Study

Neurons of the mouse were labeled with [3H]thymidine during their prenatal period of proliferation. The 3H activity of the Purkinje cell nuclei was then studied autoradiographically 8, 25, 55, and 90 days after birth. The measured grain number per nucleus decreased by about 14% between the 8th and 25th postnatal days and then remained constant up to 90 days. There was no significant decrease of the 3H activity of the Purkinje cell nuclei after correction of the measured grain number per nucleus for increasing nuclear volume of the growing Purkinje cells and for the influence of [3H]β self-absorption in the material of the sections. Injection of a high dose of [3H]thymidine into young adult mice did not result in 3H labeling of either Purkinje or other neurons in other brain regions. The results agree with the concept of metabolic stability of nuclear DNA. "Metabolic" DNA could not be observed in these experiments

In vivo cell synchrony in the L1210 mouse leukaemia studied with 5-fluorouracil or 5-fluorouracil followed by cold thymidine infusion.

[3H]-TdR and [3]-udR labelling indices and mitotic indices were followed in tumour-bearing mice after application of either 5-fluorouracil (FU) alone or of FU followed by cold TdR infusion. With FU alone, accumulation of cells at the beginning of S was found, but there was no indication of a synchronous passage of the accumulated cells further round the cycle. When FU injection was followed by cold TdR infusion, a synchronous passage of the accumulated cells through the cycle was observed. However, there was a large variation in the response of individual mice to this treatment

Automated quantitative analysis of single and double label autoradiographs

A method for the analysis of silver grain content in both single and double label autoradiographs is presented. The total grain area is calculated by counting the number of pixels at which the recorded light intensity in transmission dark field illumination exceeds a selected threshold. The calibration tests included autoradiographs with low (3H- thymidin) and high (3H-desoxyuridin) silver grain density. The results are proportional to the customary visual grain count. For the range of visibly countable grain densities in single labeled specimens, the correlation coefficient between the computed values and the visual grain counts is better than 0.96. In the first emulsion of the two emulsion layer autoradiographs of double labeled specimens (3H-14C- thymidin) the correlation coefficient is 0.919 and 0.906. The method provides a statistical correction for the background grains not due to the isotope. The possibility to record 14C tracks by shifting the focus through the second emulsion of the double labeled specimens is also demonstrated. The reported technique is essentially independent of size, shape and density of the grains