R. Allen White

Automatic processing and interpretation of DNA distributions: Comparison of several techniques

Abstract There exist a large number of apparently different methods for interpreting the distribution of DNA content in a population of cells as measured by an impulse cytophotometer (FMF). Equations are derived to provide a model of the observed numbers of cells with a given DNA content. Methods are described to compute the “ideal” distribution of cells necessary to find the number of cells in G1, S and G2 + M phase. A comparison is made of several standard methods for computing the cell age distribution. The methods are shown to be formally nearly equivalent and to give similar results when applied to an in vitro control study.

“Cytogenetic” studies of spermatids of mice carrying Cattanach's translocation by flow cytometry

The DNA content of spermatids of mice carrying Cattanachs translocation has been measured with high precision by flow cytometry. The observation that the two peaks of DNA content in the haploid region of the DNA histograms represent X-and Y-bearing spermatids was tested and confirmed. Using flow cytometry, the difference in DNA content between the X and Y chromosomes in these mice was measured to be 5.2±0.1% of the total haploid genome as compared to 3.4±0.1% in normal mice. These results demonstrated the precision of flow cytometry for cytogenetic studies. Additional information on spermatogenesis in mice bearing Cattanachs translocation was obtained and showed a gradual loss of cells during spermatogenesis in those bearing the balanced form of the translocation.

A Rapid Automated Stathmokinetic Method For Determination of In Vitro Cell Cycle Transit Times

To provide a rapid method for examining cell cycle dynamics, we utilized continuous exposure of Chinese hamster ovary cells and human colon cancer cells to colcemid to block cycling cells in metaphase, suppressing re‐entry into G1. Changes in cell cycle compartment distribution were monitored by DNA flow cytometry. Analysis of the rate of G2+ M compartment accumulation after addition of colcemid permitted calculation of all cycle transit parameters. These compared favorably with data in the same cell lines determined by the fraction of labeled mitoses technique. Serial assessment of DNA flow cytometry after addition of colcemid permits rapid quantitation of cycle traverse rates.

Assessing genetic markers of tumour progression in the context of intratumour heterogeneity.

This is a report from the Kananaskis working group on quantitative methods in tumour heterogeneity. Tumour progression is currently believed to result from genetic instability and consequent acquisition of new genetic properties in some of the tumour cells. Cross-sectional assessment of genetic markers for human tumours requires quantifiable measures of intratumour heterogeneity for each parameter or characteristic observed; the relevance of heterogeneity to tumour progression can best be ascertained by repeated assessment along a tumour progressional time line. This paper outlines experimental and analytic considerations that, with repeated use, should lead to a better understanding of tumour heterogeneity, and hence, to improvements in patient diagnosis and therapy. Four general principles were agreed upon at the Symposium: (1) the concept of heterogeneity requires a quantifiable definition so that it can be assessed repeatably; (2) the quantification of heterogeneity is necessary so that testable hypotheses may be formulated and checked to determine the degree of support from observed data; (3) it is necessary to consider (a) what is being measured, (b) what is currently measurable, and (c) what should be measured; and (4) the proposal of working models is a useful step that will assist our understanding of the origins and significance of heterogeneity in tumours. The properties of these models should then be studied so that hypotheses may be refined and validated.

A theory for the estimation of DNA synthesis rates by flow cytometry.

Abstract A method is presented for estimating the rate of DNA synthesis of a cell population by examining the DNA histogram generated by flow cytometry (FCM). The model is based on the use renewal equations to estimate the steady-state fraction of cells in each DNA compartment. The fraction of cells in each compartment is shown to be related to the Laplace transform of the transit time through that compartment. Two methods are introduced for estimating the rate of DNA synthesis utilizing different transit time distributions. One method is shown to be a simplification of the method of Dean and Anderson. The other method allows for variability in the DNA synthesis rate. The effects of quiescent cells are considered and attention is paid to the various assumptions underlying the estimation.

Cell synchrony techniques. II: Analysis of cell progression data

Abstract CHO cells which have been sorted by mitotic detachment, centrifugal elutriation and fluorescence activated cell sorting have been followed for up to 14 hr by flow cytometry to examine their progression characteristics. Mathematical modelling techniques were used to provide quantitative estimates of the cell‐cycle parameters. Mitotic detachment gives an 11.2‐hr cycle time with mean transit times TG1, Ts and TG2M equal to 3.2, 5.6 and 2.4 respectively. Cells prepared by central elutriation in an early G1 state have a 14‐hr cycle time with TG1, Ts and TG2M of 5.7, 6.0 and 2.3 hr. Populations prepared by centrifugal elutriation enriched in early S and late S and G2M have transit times of 2.7, 5.9 and 1.6 hr and 4.9, 6.7 and 2.1 hr with cycle times of 11.2 and 13.2 hr respectively. Cell sorting for a G1 population gives transit times of 9.8, 8.0 and 3.6 for an overall 21.4‐hr cycle time.

An analysis of repeated sequence heterogeneity

High-resolution thermal denaturation was used to measure the heterogeneity within repeated DNA sequences. An analysis of combined denaturation/redenaturation experiments on mouse satellite DNA suggests the existence of two minor components, one of which does not appear in the prepared EcoRII monomer. The resolving power of the denaturation/redenaturation experiment is estimated and contrasted with that of the reassociation experiment, often used to estimate repeated sequence heterogeneity. A mathematical model of the redenaturation experiment was developed and applied to mouse satellite data; the results suggest that only one-fourth of the mismatched base pairs are energetically significant in the reduction of heteroduplex stability.High-resolution thermal denaturation was used to measure the heterogeneity within repeated DNA sequences. An analysis of combined denaturation/redenaturation experiments on mouse satellite DNA suggests the existence of two minor components, one of which does not appear in the prepared EcoRII monomer. The resolving power of the denaturation/redenaturation experiment is estimated and contrasted with that of the reassociation experiment, often used to estimate repeated sequence heterogeneity. A mathematical model of the redenaturation experiment was developed and applied to mouse satellite data; the results suggest that only one-fourth of the mismatched base pairs are energetically significant in the reduction of heteroduplex stability.

State vector models of the cell cycle III: Continuous time cell cycle models☆

Abstract In this paper the elements of the matrix of the Hahn cell-cycle model are identified with the infinitesimal transition probabilities of a Markov process, and as a limiting process a differential equation analogue is derived. The probability density function of the discrete time model is derived and used to obtain the density function for transit times of the continuous time model. It is shown that the mean transit time remains constant and that the variances of the discrete and continuous time models are the same to the order of the time increment. Finally, it is shown how to derive the Takahashi model from the continuous time Hahn model.

The use of population projection matrices in cell kinetics.

Abstract Population projection matrices are applied to the study of cycling populations of cells. Simple generalizations of the method are used to study the transit time distributions through each phase: G 1 , S , G 2 and M . It is thereby possible to derive many of the common results of cell kinetics without recourse to LaPlace transforms. It is further shown that the method may be used with great ease in numerical generation of FLM curves. Two simulated curves are compared with different distributions of transit times through each phase and it is shown that the differences are experimentally undetectable.

Use of mini-microaggregated albumin® to study reticuloendothelial system (RES) function in C. parvum-treated animals☆

Mini-microaggregated albumin colloid (particle size less than 80 A) and microaggregated albumin colloid (particle size 500-1000 A) made from human serum albumin were conjugated with 99mtechnetium and used to study reticuloendothelial system (RES) function in animals treated intravenously with C. parvum. The uptake in blood, liver and spleen were studied in untreated mice and in mice at various times after treatment with 150 or 300 micrograms of C. parvum. The blood clearance of these materials was studied in rabbits before and at various times after treatment with 0.5 mg/kg of C. parvum. In mice, 80 A-microaggregated albumin was cleared significantly faster from the blood and localized to a greater extent in the liver and spleen after C. parvum treatment. There was a positive correlation between the increased organ weight induced by C. parvum and the accelerated clearance of 80 A-microaggregated albumin. The 80 A-microaggregated albumin was also cleared more rapidly from the blood of rabbits after C. parvum treatment. These studies suggest that 80 A-microaggregated albumin labeled with 99mTc is a useful tool for study of RES function in animals and because of its relatively long t 1/2 may also be useful for studies of human RES function.