IN VITRO INCORPORATION OF 3H-THYMIDINE, 3H-URIDINE, 3H-LEUCINE AND 3H-MELPHALAN BY PLASMACYTOMA PLASMA CELLS

Bone marrow plasma cells from fifteen cases of multiple myeloma, immunologically typed, were incubated with different tritiated compounds. The labelling index with tritiated thymidine is generally low, while the mean grain count is fairly normal in the active cells. The labelling index of ³H-uridine and ³H-leucine was very high, while the mean grain count per cell lies within the normal range. The results obtained with ³H-phenylalanine-mustard (melphalan), which is a drug used in the treatment of the plasmacytoma, show also incorporation values roughly comparable to those of ³H-leucine. The present data seem to support the clinical use of melphalan as a compound that is actively incorporated into the plasma cells of plasmacytoma although inhibition of protein synthesis due to specific binding to protein was not demonstrated.     

Application and efficiency of scintillation autoradiography for Drosophila polytene chromosomes

Summary A rapid method of autoradiography using the scintillation cocktail (Toluene and scintillation fluid, Omnifluor) has been described earlier. Its application and efficiency have been tested using both ³H-thymidine and ³H-uridine. The optimum time required for processing the autoradiograms has been found to be 24 h dry exposure followed by 48 h in the scintillation mixture. Detailed analysis of the autoradiograms with ³H-uridine reveals that with the rapid method the 100% level of labelling index is reached by 48 h while with the conventional method the same level is reached by 10 to 12 days of dry exposure. The maximum grain density is reached by 16 to 17 days by the conventional method. While by the rapid method, the maximum grain density is approximately 80% of the control, this grain density is reached by 48 h (plus 24 h of dry exposure) and thereafter forms a plateau. With Toluene alone the grain density never exceeds 20%. The background is also relatively low and less variable in the O-T-processed autoradiograms, as compared to the two controls. These results support that the scintillation fluid plays the key role in augmenting the labelling. Furthermore, although the maximum grain density by the rapid technique is 80% of the control, the grain density obtained by the rapid method gives less coincidence and superimposition of grains.On the other hand, with ³H-thymidine, although all labelling patterns could be resolved, the labelling index (i.e., percent of labelled cells) is about 40% at 48 h (plus 24 h) and about 79.5% at 96 h with the rapid method, as compared to about 30% and 44% with the conventional method at the two time points, respectively. Only with 16–17 days' dry exposure the ³H-thymidine labelling index increases to 67%. The frequency of the initial patterns (DD-2C) which are usually less frequent, has been found to have increased with the rapid method. No difference in grain density of labelling of ³H-thymidine could be detected between the rapid method and the conventional method. The resolution of grains also seems to be better by the rapid method, due probably to smaller size and lack of superimposition of grains. Other applications, advantages and limitations have been discussed.     

Correction of autoradiographic grain count in respect to precisely calculated background

Summary It was endeavored to find a criterion for significantly labeled cells in quantitative autoradiography. Measurements of the autoradiographic background were performed and it was found that: 1. the value of the background over the non-proliferating epithelial cells from an animal injected with ³H-thymidine is higher than over the same cells from animals not injected with an isotope, 2. the value of the background in emulsion over the tissue specimen is higher than away from the specimen. Therefore, one should take into account the background over the tissue. Nomograms are shown for quick evaluation of the percentage of cells labeled with 1, 2, 3 or 4 grains, which should be disregarded as due to the background. To obtain this percentage for a given experiment its appropriate parameters: the labeling index, the mean grain count over the cell, the standard deviation of the grain count distribution and the background grain count distribution should be taken into account.     

The circadian variations in the epithelial growth of the hamster cheek pouch: quantitative analysis of DNA distributions

The pronounced diurnal rhythm in DNA distributions of the hamster cheek pouch epithelium both in the S fraction and in the (G2 + M) fraction was compared with previous studies of the changes in tritiated thymidine labelling index and mitotic activity. The DNA distributions were obtained by flow cytometry after ultrasonic disaggregation of the isolated epithelium into a suspension of single nuclei. The DNA distributions were analysed with the computer program of J. Fried (1976) and by planimetry. The S fraction was higher than the autoradiographic labelling index during the whole 24 hr period. Only the computer fitted S fraction and the labelling index had the same difference between maximal and minimal values, and maxima at the same time of day. The DNA distributions showed a diurnal release of G1 cells into S phase proceeding through (G2 + M) phase and returning to G1 phase within a 24 hr period.     

Cell renewal in the epidermis of the loach Misgurnus anguillicaudatus (Cypriniformes)

Cell kinetics of the epidermal cells of normal juvenile loach ( Misgurnus anguillicaudatus ) were studied with autoradiography. Fish were labelled with single tritiated thymidine injections and killed at regular time intervals. Three cell types are identified by light microscopy, namely the epithelial cells, the club cells and the mucous cells. Epithelial cells are the only cell type that is involved in cell proliferation and, like the epithelial cells in the epidermis of other teleosts, proliferation of these cells occurs at all epidermal layers. The club cells and the mucous cells seem to be differentiated from the epithelial cells. Based on the time-course study of the labelling index and the grain count halving method, the generation time of the epithelial cells is estimated to be 4 days. From the labelling index of double injections, the duration of the S phase is determined as 8.3 h. Significant cell loss from the outermost layer and cell translocation from the lower layer to the upper layer within 4 days are inferred from the fluctuations of the labelling index curve. The renewal of these cells in the tissue seems rapid in comparison to the epidermis of terrestrial vertebrates.     

The Circadian Variations In the Eithelial Growth of the Hamster Cheek Pouch: Quantitative Analysis of Dna Distributions*

The pronounced diurnal rhythm in DNA distribution of the hamster check pouch epithelium both in the S fraction and in the (G₂+ M) fraction was compared with previous studies of the changes in tritiated thymidine labelling index and mitotic activity. the DNA distributions were obtained by flow cytometry after ultrasonic disaggregation of the isolated epithelium into a suspension of single nuclei. the DNA distributions were analysed with the computer program of J. Fried (1976) and by planimetry. the S fraction was higher than the autoradiographic labelling index during the whole 24 hr period. Only the computer fitted S fraction and the labelling index had the same difference between maximal and minimal values, and maxima at the same time of day. the DNA distributions showed a diurnal release of G₁ cells into S phase proceeding through (G₂+ M) phase and returning to G₁ phase within a 24 hr period.     

Developments in cell biology for quantitative immunoelectron microscopy based on thin sections: a review

Quantitative immunoelectron microscopy uses ultrathin sections and gold particle labelling to determine distributions of molecules across cell compartments. Here, we review a portfolio of new methods for comparing labelling distributions between different compartments in one study group (method 1) and between the same compartments in two or more groups (method 2). Specimen samples are selected unbiasedly and then observed and expected distributions of gold particles are estimated and compared by appropriate statistical procedures. The methods can be used to analyse gold label distributed between volume-occupying (organelle) and surface-occupying (membrane) compartments, but in method 1, membranes must be treated as organelles. With method 1, gold counts are combined with stereological estimators of compartment size to determine labelling density (LD). For volume-occupiers, LD can be expressed simply as golds per test point and, for surface-occupiers, as golds per test line intersection. Expected distributions are generated by randomly assigning gold particles to compartments and expressing observed/expected counts as a relative labelling index (RLI). Preferentially-labelled compartments are identified from their RLI values and by Chi-squared analysis of observed and expected distributions. For method 2, the raw gold particle counts distributed between compartments are simply compared across groups by contingency table and Chi-squared analysis. This identifies the main compartments responsible for the differences between group distributions. Finally, we discuss labelling efficiency (the number of gold particles per target molecule) and describe how it can be estimated for volume- or surface-occupiers by combining stereological data with biochemical determinations.     

Pattern analysis of long arm grain count as a criterion of homology in eight cases of Bp-

Summary The variation in grain count between regions of the long arms of the B group chromosomes is examined. The longer, later-labelling pair (4 s) show a different pattern of labelling to the shorter, earlier-labelling pair (5 s). It is concluded that pattern analysis may be a better criterion of homology than either length measurement or total grain count.     

Quantification of unscheduled DNA synthesis by a whole cell counting method

A procedure was developed for the quantification of the autoradiographic assay for unscheduled DNA synthesis. Relative to commonly used practices for grain counting, this procedure provides a more accurate net nuclear grain count by eliminating the subjectivity currently associated with selection of the areas to be counted for the cytoplasmic background count. Briefly, the object area and aperture area modes of an ARTEK 880 colony counter are used to collect values for the total number of silver grains over a particular cell (nuclear and cytoplasmic counts), as well as for the nuclear and cytoplasmic areas. These values are then employed in a short algorithm to determine the net nuclear grain count. This new method provides greater sensitivity for defining weak UDS responses and the data collected readily lends itself to statistical analysis.     

A computer program for analyzing bivariate flow karyotypes

This article describes a computer program for analyzing bivariate flow karyotypes of human chromosomes stained with Hoechst 33258 (HO) and chromomycin A3 (CA). The karyotype first is divided into regions that contain chromosome peaks. The chromosomes that are associated with those areas are identified. The distributions in these areas then are fitted with mathematical functions of increasing complexity. The process starts by fitting a specified number of univariate Gauss functions to projections of the HO and CA distributions of each area. The final fit can include multiple bivariate Gauss functions, including a background function for debris subtraction. The results of one stage in the fitting process serve as seed values for the next, more complex step. Since the program autonomously estimates the starting values for the iterative fitting procedures, the fit results are insensitive to operator bias and the program will consistently converge to the same solutions. The resulting table of parameter values can be used to compare flow karyotypes to a reference data set.     

Estimating kinetic parameters for single channels with simulation. A general method that resolves the missed event problem and accounts for noise.

Analysis of currents recorded from single channels is complicated by the limited time resolution (filtering) of the data which can prevent the detection of brief intervals. Although a number of approaches have been used to correct for the undetected intervals (missed events) when identifying kinetic models and estimating parameters, none of them provide a general method which takes into account the true effects of noise and limited time resolution. This paper presents such a method. The approach is to use simulated single-channel currents to incorporate the true effects of filtering and noise on missed events and interval durations. The simulated currents are then analyzed in a manner identical to that used to analyze the experimental currents. An iterative search process using likelihood comparison of two-dimensional dwell-time distributions obtained from the simulated and experimental single-channel currents then allows the most likely rate constants to be determined. The large errors and false solutions that can result from the more typically applied assumptions of no noise and an absolute dead time (idealized filtering) are excluded by the iterative simulation method, and the correlation information contained in the two-dimensional distributions should increase the ability to distinguish among different gating mechanisms. The iterative simulation method is generally applicable to channels which typically open to a single conductance level. For these channels the method places no restrictions on the proposed gating mechanism or the form of the predicted dwell-time distributions.     

THE TECHNIQUE OF LABELLED MITOSES: ANALYSIS BY AUTOMATIC CURVE-FITTING

An improved method is described for the analysis of data obtained by the technique of labelled mitoses. It is a development of the method described by Barrett (1966) in which theoretical curves are computed on the basis of a model which assumes that the phases G¹, S and G² are described by independent log-normal distributions; the analysis consists in finding a form of this model which gives a labelled mitoses curve which is the best fit to the available data. This fitting procedure has now been made automatic. No comprehensive indication of the goodness of fit can be given, although in the analysis of over fifty sets of data the method appears to have worked well.
A supplementary computer program is described which, on the basis of three separate assumed modes of cell loss, calculates the form of the age distributions and theoretical continuous labelling curves. This allows growth fraction to be calculated in a way which takes account of the distribution of phase durations and the non-rectangular age distributions of expanding cell populations. It also gives an opportunity to study the implications of continuous labelling data as regards the mode of cell loss.
A comparison is made between the present method of labelled mitoses curve analysis and the empirical rules which have often been used.     

Morphological and functional differentiation in epithelial cultures obtained from human skin explants

The present study was undertaken to characterize primary epithelial cultures obtained from human skin explants as experimental systems for studies of the differentiation process. When human skin explants were incubated at 34-35 degrees C, fibroblastic growth was strongly inhibited, whereas the epithelial growth proceeded unchanged. The lateral growth of the epithelial cells could be divided into two phases - a migratory and a proliferative one. Only cultures incubated at 35 degrees C or below completed the morphological differentiation process before sloughing, whereas no qualitative difference in protein synthesis was observed between cultures incubated at temperatures from 33-37 degrees C. Cultured epidermal cells were labelled with 3H-thymidine and analysed by flow cytometry and cell sorting. Cells sorted from the S- and G2-phase populations were further analysed by autoradiography and a considerable heterogeneity as to the nuclear labelling was disclosed. A large fraction of S-phase cells were found to be totally unlabelled. The grain count distributions revealed similar cell cycle subpopulations as have been shown to occur in vivo. The relationship of these subpopulations to the differentiation process is discussed.     

The decay of autoradiographic grain number over crypt base columnar cells in murine ileum as a measure of their generation time

The decay in the number of grains over [3H]-thymidine labelled crypt base columnar cells (BCC) in autoradiographs of the ileum of BDF1 mice has been studied. The results revealed that using the conventional grain count halving (GCH) method it is possible to obtain an estimation of the generation time (Tc) of the proliferative BCC cells in the Paneth cell zone (PC-zone) of 18.8 +/- 0.74 h. This lies within the range obtained by the percent labelled mitoses (PLM) method, but is shorter than most values obtained by stathmokinetic methods. The present data show no evidence for a shortening of the cell cycle 3 days after irradiation (8 Gy) which is contrary to some earlier observations. Some reasons for this discrepancy are discussed. The comparatively high labelling index of the BCC allows a larger amount of data to be easily collected, compared with the PLM technique, and correction factors which take into account the complicated shape of the bottom of the crypt are not required.     

An improved procedure for background correction in autoradiography

In the event of weak autoradiographic labelling, the proportion of truly labelled cells or structures can be calculated from the frequency distributions of grains per area or cell structure for i = 0, 1,..., n grains using the results obtained for an experimental group after the application of a radioactively labelled substance and those obtained for a control group without radioactivity. The principle of this computer-aided method is also applicable when the grain counts are related to varying areas in histological sections.     

KINETICS OF CELL RENEWAL, CELL MIGRATION AND CELL LOSS IN THE HAIRLESS MOUSE DORSAL EPIDERMIS

Forty hairless mice were given injections of tritiated thymidine every 4th hour during 10 days. At 24 hr intervals groups of four mice were killed. The numbers of labelled basal and differentiating cells were determined by autoradiography with a stripping film technique. To determine the background activity skin sections from uninjected control mice were subjected to the same stripping film procedure. Another group of hairless mice was given one single pulse labelling with tritiated thymidine. The number of labelled mitoses was scored for 12 hr after the injection. At 10, 12 and 15 hr after the injection, the numbers of labelled basal and differentiating cells were also determined. A mathematical model of cell population kinetics in the epidermis has been suggested. The results of different simulations on this model were compared with the observed results. The curve of mean grain counts under continuous labelling increased from day to day with two well-defined plateaux. The percentage of all labelled cells increased rapidly up to the 3rd day, and thereafter the curves gradually flattened off. When basal cells and differentiated cells were considered separately the labelling index of the basal cells increased rapidly for the first 3 days and then flattened off at the 100% level on the 5th day. The labelling index of the differentiating cells was low during the first 3–4 days. Then a steep increase in the percentage of labelled differentiating cells was seen, but the curve flattened off again close to the 100 % level after the 7th day. The labelled mitosis curve had its maximum 5 hr after the thymidine injection. The curve fell again to almost zero at 12 hr. Ten, 12 and 15 hr after the injection, 6, 7 and 7% respectively of the labelled cells were found in the spinous layer. It was concluded that three grains over each nucleus could be used as lower limit for considering a cell as labelled. On this basis, tritiated thymidine injections every 4th hour can be considered as continuous labelling.     

The kinetic significance of cell size : II. Size distributions of resting and proliferating cells during interphase

This second part in a two part report describes the kinetic, cell size and nuclear size characteristics of S phase cells and cells with greatly protracted generation times (‘resting’ cells) in a cell line of human lymphoid cells. The median cell and nuclear sizes of S phase cells were greater than the corresponding median sizes observed in the whole population. Resting cells (operationally defined as unlabelled cells after 5 days of continuous labelling with [³H]TdR) have cell and nuclear size distributions overlapping with the cell and nuclear size distributions of the whole population. These resting cells are kinetically characterized by means of the observed labelling index vs time data during continuous labelling. The implication of these results are discussed.     

Epidermal chalones and squamous cell carcinomas. The growth inhibitory effects of aqueous epidermal extracts (G1 and G2 chalones) on the epidermis and on a transplantable keratinizing carcinoman in nude mice.

Balb/c/nu nude mice that had been transplanted with a moderately differentiated squamous cell carcinoma were injected i.p. with different doses of epidermal chalone, and control animals were injected with saline. The labelling indices (H3TdR) and the mitotic rate (stathmokinetic method with vinblastine sulphate) were determined. In the untreated animals, both the labelling index and the mitotic rate of the tumor were considerably higher than in the epidermis, and the rate of cell birth was almost twice that of the epidermis. Higher doses of chalone were needed to reduce the labelling index for the tumour than for the epidermis, and there was generally a less pronounced dose/response relationship in the tumours than in the epidermis. The same was true of the mitotic rate but here the results were not as obvious as for the labelling index. A possible explanation of the results may be that the tumour cells are less sensitive than epidermal cells to the injected chalones, or that reduced vascularization of the transplanted tumour may lead to reduced access of chalone, or that tumour necrosis may pay a role. However, it is evident that the tumour cells react less than the epidermis to both the G1 and the G2 chalone, and thus the findings of this study do not provide any evidence against the theory that epidermoid transplanted tumours are less sensitive to epidermal chalones than normal tissue of the same histogenetic origin.     

Method of binary classification and risk assessment of individuals with familial polyposis based on [3H]TdR labelling of epithelial cells in colonic crypts

An analysis has been developed to improve the quantitation of abnormal patterns of tritiated thymidine [(3H]TdR) labelling of colonic epithelial cells, in biopsy specimens removed from human subjects at varying degrees of risk for colon cancer. After pulse incubation of specimens of colonic mucosa with [3H]TdR, each subject's microautoradiographic epithelial cell labelling distribution was segregated into eleven compartments over entire colonic crypts. The findings of each subject were then analysed to determine their relative degree of similarity to the findings for two reference populations of interest, i.e. a high-risk and a low-risk population; the individual was then classified as being closer to one or the other of the reference populations. The analysis developed is based upon a comparison of multinomial probabilities for the distributions of the labelled cells within the crypts, and permits the routine categorization of uneven distributions of labelled cells. For each subject, certain linear scores, a prognostic index based on them, and a related presumptive risk, were calculated. The sensitivity with which individuals known to be symptomatic for polyposis, and the specificity with which individuals known to be at lower risk were determined, were 73 and 93% respectively. The results suggest that this method of distinguishing among integer distributions of [3H]TdR- labelled cells in biopsies of colonic mucosa, may provide a useful basis for identifying individuals with familial polyposis, by separating their labelling patterns from those of low-risk subjects.     

An improved procedure for background correction in autoradiography

Summary In the event of weak autoradiographic labelling, the proportion of truly labelled cells or structures can be calculated from the frequency distributions of grains per area or cell structure fori=0, 1,...,n grains using the results obtained for an experimental group after the application of a radioactively labelled substance and those obtained for a control group without radioactivity. The principle of this computer-aided method is also applicable when the grain counts are related to varying areas in histological sections.Dedicated to Professor Dr. T.H.Schiebler on the occasion of his 65th birthday