Aspects of statistics in studies of cell proliferation. I. Multistage sampling

In cell kinetic experiments, as in many other branches of biological science, we often sample at various stages of an experiment: we may for example take a sample of animals, then from each study a sample of sites, and from each site take replicate observations. This sampling process can be optimized to give maximum precision to an estimated quantity, but care must be taken in analysing data so gathered because the analysis depends on the precise sampling strategy.     

Measuring the relative information in allele-sharing linkage studies

In the context of allele-sharing methods, this article investigates ways of measuring the information in the marker data relative to the amount of information that would have been available if the identity-by-descent (IBD) process were known. Such measures are needed to decide whether new markers can substantially modify the evidence for excess sharing. We propose new measures that take advantage of the properties of the exponential model introduced by Kong and Cox (1997, American Journal of Human Genetics61, 1179-1188). These measures are related to Fisher Information and hence are also efficiency measures. Large-sample and small-sample properties of the new and previously proposed measures of information are examined.     

Stimulation of rat pituitary tumoral cell proliferation in vitro by tetra- and pentagastrin.

The effects of the active gastrin fragments, penta- and tetragastrin on the incorporation of tritiated thymidine into the rat pituitary tumoral cells were investigated in vitro. The significant stimulation of 3H-thymidine incorporation was observed as an effect of the investigated substances. The putative role of gastrin as an autocrine growth factor active in pituitary tumorigenesis was discussed.     

Immunocytochemical analysis of chromaffin cell proliferation in vitro.

The bromodeoxyuridine (BrdU) incorporation technique for immunocytochemical labeling of S-phase nuclei was optimized for the study of chromaffin cell proliferation. Sequential fixation in ethanol followed by paraformaldehyde, and the use of DNAse to render incorporated BrdU accessible to antibody, permitted permanent double staining for BrdU and tyrosine hydroxylase. The efficacy of the technique was demonstrated in microcultures of dissociated neonatal rat adrenal glands, in which chromaffin cells exhibited proliferative responses to nerve growth factor and fibroblast growth factor similar to those previously demonstrated by autoradiography. Growth factor responsiveness was observed in both serum-containing and serum-free medium.     

In vitro studies of the effect of intermittent compressive forces on cartilage cell proliferation.

Intermittent compressive (IC) forces (96 mm Hg, 0.3 Hz) inhibit by 35–60% the serum stimulated increase in ornithine decarboxylase activity (ODC) in chick embryo epiphyseal cartilage cells and rat chondrosarcoma cells. IC had no effect on mouse fibroblast L-cells ODC. The dose-response pattern of the IC effect indicated an all-or-none response with a threshold at 80 mm Hg, a pressure roughly equivalent to the in vivo weight bearing force. The k_m of the cartilage cell ODC, measured at four hours, was about 0.1 mM and was not affected by IC. The V_max, on the other hand, was significantly reduced by IC which is consistent with less enzyme or non-competitive inhibition. IC also produced a significant increase in cAMP levels in both cartilage explants and isolated cells in the presence and absence of serum and a significant reduction in ³H-thymidine incorporation into DNA. The findings show that cellular cAMP, on one hand, and ODC and DNA synthesis, on the other hand, change in opposite directions following exposure to serum and/or IC. Investigation of the IC effect on DNA synthesis in serum-deprived synchronized cartilage cells revealed that IC reduced the number of cells going into S but did not lengthen the G₁ phase. Exposure to IC early in G₁ (0–13 hours) produced the full effect, whereas IC application between 13 to 24 hours (pre S) had no effect. IC had no effect on ³H-thymidine incorporation in L-cells.     

EGF and TGF-alpha stimulate retinal neuroepithelial cell proliferation in vitro.

Peptide growth factors have been shown to have diverse effects on cells of the CNS, such as promoting neuronal survival, neurite outgrowth, and several other aspects of neuronal differentiation. In addition, some of these factors have been shown to be mitogenic for particular classes of glial cells within the brain and optic nerve, and recently two peptide growth factors, fibroblast growth factor and nerve growth factor, have been shown to have mitogenic activity on the CNS neuronal progenitors. We now report that two members of another peptide growth factor, epidermal growth factor and transforming growth factor-alpha, are mitogenic for retinal neuroepithelial cells in primary cultures and provide evidence for the presence of both of these factors in normal developing rat retina.     

Cytogenetic studies of three triazine herbicides. I. In vitro studies

Atrazine, simazine, and cyanazine are widely used pre-emergence and post-emergence triazine herbicides that have made their way into the potable water supply of many agricultural communities. Because of this and the prevalence of contradictory cytogenetic studies in the literature on atrazine, simazine, and cyanazine, a series of in vitro experiments was performed to investigate the ability of these three triazines to induce sister chromatid exchanges (SCEs) and chromosome aberrations (CAs) in human lymphocyte cultures. Our results showed that all three triazines failed to produce any significant increases in SCEs or CAs up to the limits of solubility [using 0.5% dimethyl sulfoxide (DMSO)]. Our results are discussed in light of contradictory results in the literature.     

Intestinal cell proliferation. I. A comprehensive model of steady-state proliferation in the crypt

Cell replacement in the crypt of the murine small intestine has been studied and modelled mathematically under steady-state conditions. A great deal of information is available for this system, e.g. cell cycle times, S phase durations, the rate of daily cell production, the Paneth cell distribution etc. The purpose of the present work was to consider simultaneously as much of these data as possible and to formulate a model based upon the behaviour of individual cells which adequately accounted for them. A simple mathematical representation of the crypt has been developed. This consists of sixteen stem cells per crypt (TC = 16 hr, TS = 9 hr), and four subsequent transit cell divisions (TC = 11 to 12 hr, TS = 8 hr) before maturation. Experimental data considered to test the modelling were LI and data on the number of vertical runs of similarly labelled cells. All data were obtained from the ileum after 25 microCi [3H]TdR given at 09:00 hours. A number of alternative assumptions have been considered and either accepted or rejected. Two alternative model concepts of cell displacement explain the data equally well. One is dependent upon strong local cell generation age determinance while the other could accommodate any weak local cell displacement process in conjunction with an environmental cut-off determinant at the middle of the crypt. Both models provide new interpretations of the data, e.g. certain rates of lateral cell exchange between neighbouring columns (250 to 350 per crypt per day out of a total of 420 cell divisions per day) can be concluded from run data, while LI data provide information about the mechanisms involved in maintaining a position-related age order in the crypt.     

Functional studies on B cell hybridomas with B cell surface antigens. I. Effects of anti-immunoglobulin antibodies on proliferation and differentiation

B cell hybridomas with Ia and IgM molecules on the cell membrane were treated with either purified goat anti-mouse mu antibody (anti-mu) or monoclonal rat anti-mouse IgM antibody (anti-IgM). The spontaneous uptake of [3H] thymidine by these cells was markedly inhibited by both reagents. These hybrid cells could be induced to differentiate into IgM-secreting cells in the presence of these reagents at high frequency. Furthermore, the induction of IgM secretion by B cell hybridomas treated with these antibodies was completely T cell independent, and cell division was not required for the differentiative response to anti-mu. In addition, F(ab')2 fragments of anti-mu showed more effects on proliferation and differentiation of these cells than intact anti-mu. Interestingly, TH2.54, a subline of B cell hybridomas, could generate IgG2a production as well as IgM when incubated with anti-mu. These findings suggest very strongly that the interaction of either goat anti-mu or monoclonal rat anti-IgM with surface IgM molecules on the cell membrane of the B cell hybridomas inhibits in vitro spontaneous proliferation, and results in providing signals for differentiation into Ig-secreting cells without T cell factors.     

In vitro effects of a new fused azaisocytosine-like congener on relative cell proliferation,necrosis and cell cycle in cancer and normal cell cultures

Molecular and Cellular Biochemistry - The study was aimed at describing the mode of action of an innovative drug-like congener of fused azaisocytosine—EIMTC (ethyl...     

In vitro secondary MLR. I. Kinetics of proliferation and specificity of in vitro primed responder cells.

We have examined the kinetics and specificity of secondary in vitro mixed lymphocyte reactions (MLR). With limited numbers of primed responder cells (PRC) in the presence of "excess antigen" it was possible to obtain proliferative responses that were proportional to the number of PRC initially placed in culture. The responding cells, after an initial lag period, seem to grow exponentially until day 3 of culture. The responses of PRC (with the strain combinations and culture conditions described in this report) seemed to be directed toward stimulator cell determinants whose expression was determined by genes in the I region of the MHC. In one case, the relevant incompatibilities could be further restricted to the I-A region. Although PRC responded best to stimulator cells sharing the I region with the priming stimulator cell, apparent cross-reactivity could be observed by restimulating PRC with stimulator cells that did not carry the MHC haplotype of the priming stimulator cell. The rate of proliferation (measured as 3H-thymidine incorporation) in these apparent cross-reactions was reproducible and comparable to the rate observed in response to the priming stimulator cell. It was possible, therefore, to estimate the proportion of PRC that reacted in the presence of third party stimulator cells compared to the response of these PRC to the priming stimulator cells. We have estimated that the response of A (B6) PRC against H-2d and H-2s haplotype stimulator cells is about half of the response of these PRC to H-2b, the priming stimulator cell.     

Aspects of statistics in studies of cell proliferation. IV. Simulation

Computer simulation of an experiment can help to reveal how effective that experiment might be in estimating cell kinetic parameters, or in elucidating the nature of a proliferative response to a perturbation of the steady state, such as a carcinogenic or therapeutic treatment. Writing a simulation also clarifies one's thoughts about possible mechanisms of the proliferative response.     

In vitro studies on transplantation immunity. I. MIF production by sensitive lymphocytes in mice

Sensitized lymphocytes from mice immunized with skin homografts produce migration inhibitory factor upon incubation with lymphocytes (antigen) from the sensitizing strain. The MIF is produced within 14 hr following incubation of sensitized lymphocytes and antigen. In this reaction, antigenic specificity is a prerequisite for MIF production; however, the action of MIF transcends the strain barrier. Also, MIF produced in homograft reactions in mice inhibited the migration of peritoneal cells from normal guinea pigs. Finally, lymphocytes from mice bearing skin homografts do not develop the capacity to produce MIF prior to the rejection of the sensitizing skin grafts.