Labelled Mitoses Method Research Articles

An Overview of Models of Cell Proliferation

Some different types of models of cell proliferation are discussed. The importance of basing the models on experimental data is emphasised, but warnings are given about some of the pitfalls in fitting models of data. The importance of investigating alternative models which might lead to similar experimental findings is stressed. The use of simulation to assess the ability of an analytical method to extract correct information from experimental data is advocated. In this instance, the modelling process takes place in advance of the data collection. Models described relate to cell proliferation in a transplantable tumor, the prostate of the castrate mouse stimulated with testosterone, and stratified squamous epithelium. Experimental techiques include measurment of tumor size, calculation of labelling and mitotic indices over time, and the fraction labelled mitoses method.

The effect of the slow-to-green mutation on cell division during leaf initiation and early leaf growth in Lolium temulentum.

Cell division was examined during leaf initiation in the slow-to-green mutant of Lolium temulentum L. to test the hypothesis that the cell cycle in the leaf primordium is a key regulator of the well characterized reduction in final leaf length in the mutant compared with that of the wild type. The cell doubling time (cdt, by colchicine method) was substantially longer in the youngest leaf primordium (YLP) of the mutant (107 h) than in the wild type (43 h) although the duration of the most rapid cell cycle (cc, by percentage labelled mitoses method) was between 18-20 h in each. As a consequence, the proportion of rapidly proliferating cells was only 20 % in the mutant compared with 47 % in the wild type. The size of the shoot apical meristem and the plastochron were similar between genotypes which indicates that the shoot meristem was largely buffered from the effects of the mutation. Mitotic cell area was also similar in the YLP of both genotypes. However, as the leaf elongated, mitotic cell area and interphase cell size were significantly larger in the mutant compared with the wild type. This change was coupled with a reduced number of cells per unit length of leaf in the mutant. The data are consistent in showing that the proportion of rapidly proliferating cells in the YLP (but not the rate of cell division) is a key parameter which influences growth of the leaf.

Detection of the Ki-67 antigen in fixed and wax-embedded sections with the monoclonal antibody MIB1.

Novel antibodies have been generated by immunizing with bacterially expressed fragments of the repetitive motif of the Ki-67 gene. One such antibody, MIB1, recognizes a fixation and embedding resistant epitope on the Ki-67 protein if sections are previously microwaved in a citrate buffer. We have investigated the utility of this antibody as a marker of cell proliferation in archival material. The microwave technique is simple but requires careful monitoring since different tissues and fixatives require different irradiation times. Strong nuclear immunoreactivity was detected with all fixatives studied. Cytoplasmic staining was not identified. In a wide range of normal tissues the distribution and number of MIB1 immunoreactive cells matched that of cryostat sections stained with Ki-67. In nude mouse xenografts in which the growth fraction had been defined using a fraction of labelled mitosis method, the labelling index with MIB1 matched that previously determined for Ki-67 and correlated well with the growth fraction. Other markers of proliferation (e.g. proliferating cell nuclear antigen) have been shown to be expressed in DNA repair, thus we investigated expression of MIB1 immunoreactivity in situations of DNA repair in vivo--ultraviolet irradiated human skin. MIB1 staining correlated with semi-conservative DNA synthesis rather than excision repair DNA synthesis. Finally, the morphological and cell cycle distribution of MIB1 expression is identical to that of Ki-67. Thus, MIB1 represents a new anti-Ki-67 antibody which appears to be a robust marker of cell proliferation easily applicable to archival material.

A comparison of immunohistochemical markers of cell proliferation with experimentally determined growth fraction

The relationship between immunoreactivity for cell proliferation markers (Ki67 and PCNA) and the growth fraction as determined by the fraction of labelled mitoses method was assessed in xenograft tumours grown from the LoVo cell line in nude mice. FLM curves were constructed by injecting tritiated thymidine and then preparing autoradiographs from the tumours. From this data an estimate of growth fraction and cell cycle time were made. Using frozen material from the same tumours, the Ki67 index was determined by immunostaining. PCNA staining was determined in the fixed material which had been used for the autoradiographs. The results show that Ki67 staining follows the same trend as the FLM-determined growth fraction as the tumour increases in size and the rate of growth decreases. However the Ki67 index does produce a consistent overestimate of the growth fraction in this in-vivo system, as compared to observations in cell culture. PCNA staining showed virtually 100 per cent positive staining in all the tumours, which is likely to reflect the long half-life of the antigen, compared to the very fast cell-cycle time of the xenograft tumours. These results show that staining with proliferation markers is not a precise determinant of growth fraction. Ki67 staining is a method that can be usefully applied as an operational marker of cell proliferation, but should not be used uncritically. Further caution is necessary in the use of PCNA. The findings also demonstrate the need to use a range of methods when assessing a new proliferation marker.

Effect of two aliphatic aldehydes, methylglyoxal and 4-hydroxypentenal, on the growth of Yoshida ascites hepatoma AH-130

The influence of a ketoaldehyde, methylglyoxal (MG), and a hydroxyalkenal, 4-hydroxypentenal (HPE), on the growth of a highly-deviated tumour has been investigated. MG and HPE, administered intraperitoneally, strongly depressed in rats the proliferative activity of the Yoshida ascites hepatoma AH-130, reducing its mitotic and labelling indices as well as the proportion of cycling cells (growth fraction). Monitoring the effects on the cell cycle by the labelled mitoses method showed that the percentage of labelled mitoses was markedly lowered after either aldehyde, which is indicative for a blocking effect in the S phase. In addition, the mean cell cycle time was slightly prolonged by MG, probably due to accumulation of cells in G1, whereas HPE delayed the first mitotic peak and increased the mean DNA synthetic period without modifying the overall cycle time. The effects of HPE on the cell cycle were prevented by pretreatment with polyamines. Repeated doses of MG significantly increased the fraction of tumour-bearing rats surviving at 90 days (‘indefinite’ survivors) as well as the survival time of those which succumbed, implying that the carcinostatic effect of MG persisted over several cell cycles. By contrast, HPE did not significantly modify the survival of AH-130-bearing rats, suggesting that its influence on tumour growth was rapidly reversible.

Visualization of chromosome assembly during the S and G2 stages of the cycle and chromosome disassembly during the G1 stage in semithin sections of mouse duodenal crypt cells and other cells.

Previous examination of dividing cells in the isthmus of the mouse pyloric antrum by using semithin (0.5-micron-thick) Epon sections revealed that the prophasic condensation of chromosomes began early in the DNA-synthesizing (S) stage. In order to examine whether the same observation could be made in other proliferating cell types, the crypt base columnar cells in mouse duodenum and the hepatocytes of the rat 48 hr after partial hepatectomy were investigated by morphologic and radioautographic techniques. When crypt base columnar cells were studied in semithin Epon sections, the four phases of mitosis showed the characteristic features described by classical cytologists. Moreover, the proportion of cells in prophase and telophase was high. To relate the mitotic phases to the stages of the cell cycle, the "frequency of labeled mitoses method" provided the duration of the cell cycle, 12.3 hr, and of the S stage, 7.3 hr. From the frequency of the occurrence of mitotic phases, it was estimated that metaphase lasted 0.3 hr and anaphase 0.11 hr, in line with previous estimates. However, the durations of prophase and telophase were long, 5.9 and 1.9 hr, respectively. The whole mitotic process took over 8 hr. From the duration of prophase and cycle stages, it was calculated that 67% of the S stage was occupied by prophasic cells. In fair agreement with this estimate, 68% of the labeled cells 10 min after a 3H-thymidine injection were found to be in prophase. In regenerating hepatocytes, the morphological features and frequency of prophase and telophase cells were similar to those observed in duodenal crypt cells. While the cycle time was not measured and, therefore, the duration of cycle stages and mitotic phases could not be estimated, it is likely that their duration would be of the same order of magnitude. In conclusion, the mitotic process in duodenal crypt cells takes over 8 hr. Moreover, the crypt cells, like antral isthmal cells, show features of early prophase soon after they enter the S stage of the cycle.

A modification of the grain count halving method for detailed analysis of cell kinetic parameters.

A modification of the conventional grain count halving (GCH) method is presented. By determining the decrease of the mean grain number of all interphase cells in addition to that of all labelled interphase cells on the same autoradiographs, the potential doubling time Tpot (or the cell production rate kp) can be obtained in one and the same experiment. Thus the modified GCH method provides not only the cycle time of the cell population studied but also the growth fraction and, with additional cytofluorometric measurements, the duration of all cycle phases. By evaluating the cell production rate and the growth fraction this method leads to more reliable cell kinetic data of experimental tumours and human tumours growing in nude mice. In contrast to other cell kinetic methods, the modified GCH method can also be applied in special cases to human tumours in vivo, since only few points of measurement are needed. A comparison of the cell kinetic results obtained by the modified GCH method with those derived from the fraction of labelled mitoses method, both applied to allotransplants of the adenocarcinoma EO 771 in nude mice, shows good agreement.

Effects of interpolated dark periods during the first long day of floral induction on the cell cycle in the shoot apex of Silene coelirosa

Seeds of Silene coeli‐rosa L. were germinated and grown at 20°C in short days of 8 h light from fluorescent and tungsten (F + T) bulbs and 16 h darkness for 28 days (day 0). At 1700 h of day 0, the plants were exposed to 16 h light from T (LD) followed by 8 h F + T, or the same treatment interrupted at 1700 h of day 0 by 20 or 60 min darkness. Plants were exposed to tritiated (methyl‐[3H])‐thymidine for 2 h (1645–1845 h) and sampled every 2 h for 24 h. The cell cycle (percentage labelled mitoses method), and changes in cell number were measured in the shoot apical meristems. The cell cycle in the LD, 20 and 60 min dark‐interrupted LD (diLD) treatments was 10, 11 and 13 h, respectively. Mean cell generation times were ca 3–5 h longer, suggesting that the shorter cell cycles were transient. The proportions of cells with 2C or 4C amounts of nuclear DNA, indicated that imposition of darkness resulted in a progressive lengthening of G1 from about 3 h in the LD to 7 h in the 60 min diLD treatment. Conversely, G2 shortened from about 4 h in the LD to 3 h in the 60 min diLD treatment. Measurements of labelling index indicated that S‐phase was about 1.5 to 2 h in each treatment. The data are discussed in relation to the known inhibitory effect of the diLD treatments on flowering.

Influence of the beta-adrenergic antagonists propranolol, practolol and oxprenolol on the proliferation of rat jejunal crypt cells.

Beta-adrenergic blockade by quite large doses of propranolol, practolol and oxprenolol, once or continuously applied, does not influence jejunal crypt-cell proliferation in the rat. After a single i.p. injection of 20 mg/kg propranolol or practolol and even of 100 mg/kg practolol, the mitotic index, the labelling index and the duration of the S phase do not differ between treated and untreated control animals nor between animals treated with the different drugs. Continuous application of 30 mg/kg/d propranolol, practolol or oxprenolol for 7 or 14 days does not affect the mitotic and labelling indices either, nor does it change the duration of the cycle of the jejunal crypt cells and its phases as determined by the percent labelled mitoses method. These results are in contrast to those reported previously by Tutton & Helme (1974).

Estimation of circadian variations in cell cycle phase durations in murine epidermal basal cells.

Circadian variations in the proliferative activity of squamous epithelia are well known. However, circadian variations in the duration of the various cell cycle phases (S, G2 and mitosis) have been disputed. The percent labelled mitoses method, which is traditionally used to obtain duration of cell cycle phases, is poorly suited for identification of circadian variations. Therefore methods combining changes in compartment size (cell cycle phase) and cellular flux through the compartments have been used. Three different methods using such data are presented. These incorporate various simplifying assumptions that cause methodological errors. Limits for use of the different methods are indicated. The use of all three methods gives comparable and pronounced circadian variations in the duration of S and G2 phase. These results are also compatible with circadian variations in the mitotic duration, but they may also represent artefacts due to sensitivity to model errors.

Non-circadian periodicity in the duration of the cell cycle and the G1 phase in the hindlimb epidermis of the anuran tadpole, Rana pipiens.

Using the percentage labeled mitoses method, seven cell cycle determinations were initiated at 6-hr intervals over a 36-hr span in order to see if the cell cycle in the tadpole hindlimb epidermis varied with time or showed rhythmicity. There was a pattern of two long cell cycles followed by a shorter one. Total cell cycle length (Tc) and the length of the G1 phase plus one-half of the mitotic time (TG1 + 1/2M) fluctuated the most, although only TG1 + 1/2M varied significantly with the Chi-square test. The proportion of TC spent in each phase was also calculated. Only TG1 + 1/2M/TC had statistically significant fluctuations with time. Rhythmicity was analyzed by a computer program using the method of least squares for cosine curve fitting. Statistically significant ultradian rhythms of 18.4 hr in TC, 18.5 hr in TG1 + 1/2M and 18.6 hr in TG1 + 1/2M/TC and the length of the DNA synthetic phase/total cell cycle length (TS/TC) were found. Circadian rhythmicity was not observed. The acrophases of the ultradian rhythms of TC and TG1 + 1/2M coincided, suggesting that the rhythm of TC was due mainly to variation in TG1 + 1/2M. In the absence of significant variation in TS, the longest phase of the cell cycle, whenever G1 + 1/2M was short, TS/TC increased, so that the 18.6 hr rhythm in TS/TC was also a result of the periodicity in TG1 + 1/2M.

The effect of demonstration of the Brazelton neonatal, assessment scale on the father infant relationship

Acute granulocytic hyperplasia was produced in mice by injection of endotoxin and, using the labelled mitosis method, the duration of DNA synthesis was compared to that in mormal mice. No difference was found. Taken in conjunction with previous studies using labelling indices, the results indicate that in mice showing acute granulocytic hyperplasia, there is only a moderate increase in proliferation of mitotable granulocytes. Thus the major factor responsible for hyperplasia is increased differentiation of precursor cells.

Kinetics of in vitro ageing of mouse embryo fibroblasts.

The kinetics of ageing of normal mouse embryo fibroblast cells in culture have been determined. The growth pattern during every passage was established. It was observed that the growth pattern was not exponential, but that the growth rate declined progressively both within and with every passage. We also estimated the cell cycle parameters using the Fraction of Labelled Mitoses method at every passage. We found that the cell cycle duration was constant throughout the lifespan of this cell strain; the median value of the cell cycle duration was found to be 15.5 +/- 0.5 h (S.D., n = 8). From these two sets of observations we infer that the fraction of dividing cells declines smoothly from the beginning of the culture. Our data exclude quite positively any description of ageing of the fibroblast population in terms of a catastrophe or any abrupt change in the population. Our data are also inconsistent with a linear decline in growth fraction. On the contrary, we observed that there was a gradual and smooth decline in the growth rate of the strain, due to a smoothly declining growth fraction. This smooth change in the growth behaviour of this cell strain is accurately described by the mortalization theory of Shall & Stein in which the single parameter gamma (gamma), describes the change in reproductive potential over the entire lifespan. The parameter gamma describes the rate at which the doubling time of the culture increases. It is the number of generations at which half of the newborn cells are themselves reproductively sterile. Our present data provided an estimate of gamma for this cell strain, which was consistent during the entire lifespan of the strain; the best estimate of gamma for this cell strain was 20.3 +/- 0.6 generations (S.D., n = 19).

Stage specificity in the mesenchyme requirement of rodent lung epithelium in vitro : a matter of growth control?

Epithelia from lung rudiments in which secondary bronchial buds are already established (14th and 13th gestational day for rat and mouse respectively) are able to undergo branching morphogenesis and cytodifferentiation in submandibular mesenchyme in vitro, whereas lung epithelium from one day younger foetuses rarely gives a morphogenetic response to submandibular mesenchyme and usually differentiates into primary (non-budding) bronchial epithelium. The failure of 13-day rat lung epithelium to respond to submandibular mesenchyme can be prevented by peeling off the submandibular mesenchyme from the lung epithelium after 2 1/2 days culture and replacing the same mesenchyme, or renewing it with fresh salivary mesenchyme ex vivo. Changes in the epithelial contour are visible by 10 h and buds form within 24 h; this is followed by branching morphogenesis in more than 66% of the samples. The number of cells in S-phase in the epithelium is doubled within 3 to 5 h after the operation and the number of mitotic cells (colchicine block) is increased during an 11 to 19 h period after the operation. Substituting stomach mesenchyme for submandibular mesenchyme after the operation failed to elicit morphogenesis or an increase in the number of S-phase cells in the epithelium. The proportion of epithelial cells in S-phase in unoperated recombinants does not differ from the proportion in the primary bronchial epithelium (non-budding) of homotypic lung recombinants, whereas the proportion of S-phase cells in operated recombinants approaches that found in the buds of homotypic lung recombinants. The distribution of S-phase cells in visibly responding recombinants 15 to 17 h after operation shows the same heterogeneity as in homotypic lung recombinants, newly formed buds having twice as many cells labelled with [3H]thymidine as the non-budding area. Cell cycle parameters of intact rat lung growing in vitro were estimated using the labelled mitoses method. Primary bronchial epithelium and bronchial buds both had a total cell cycle time of about 13 h and an S-phase of about 10 h. The growth fraction was 0.54 in the primary bronchus and 0.95 in the buds. It is suggested that, also in the recombinants, differences in the proportion of S-phase cells at any one time in morphogenetically active and inactive areas of the epithelium are due to differences in the growth fraction. It is concluded that an early event in the morphogenetic response of lung epithelium to submandibular mesenchyme after removing and restoring the mesenchyme is an increase in the size of the population of dividing cells and it is suggested that a high proportion of dividing cells in an epithelial population is a prerequisite for further interaction of epithelium and mesenchyme leading to branching morphogenesis.

Simian virus 40 A gene function: further characterization and growth of tsA transformed chinese hamster cells.

Chinese hamster embryo cells transformed with the tsA 58 mutant of Simian virus 40 express the transformed phenotype at the permissive temperature (33 degrees C or 37 degrees C) and a "normal" phenotype at the nonpermissive temperature (40.5 degrees C). Immunofluorescence and immunoprecipitation of T antigens demonstrated that the "T" antigen (100 K) has an increase rate of synthesis and degradation at 40.5 degrees C. However, the cells continue to replicate at the nonpermissive temperature when assayed by flow cytometry and autoradiography. This DNA synthesis was cellular, not viral, and not owing to an increase in DNA repair. When the cell cycle distributions of G1, S, and G2 + M were assayed by the fraction labeled mitoses method, no differences were evident at the permissive and nonpermissive temperature; however, the doubling time was lengthened at 40.5 degrees C (13 hours vs. 100 hours). These results suggest that at 40.5 degrees C, the tsA transformed cells are cycling and dying. However, if the transformed cells are seeded onto monolayers of normal Chinese hamster cells at 40.5 degrees C, the cells are growth arrested when measured by growth assays, flow cytometry, autoradiography, and immunofluorescence for T antigen. Therefore, growth arrest can be obtained in tsA 58 transformed Chinese hamster cells when cocultured with normal Chinese hamster cells.

Cell cycle kinetics of regenerating urothelial cells of the rat urinary bladder after partial cystectomy.

The cell cycle kinetics of bladder urothelial cells regenerating after partial cystectomy were investigated in 96 female Wistar rats using the percentage labelled mitoses method. In the area of resection a mean cell cycle time (TC) of 15 h was determined. The DNA synthesis phase (TS) lasted 6 h and the premitotic-postsynthetic phase together with the mitosis phase (TG2 + M) 1.5 h, thus giving a presynthetic-postmiotic phase (TG1) of 7.5 h. Similar values were found for the urothelial cells in the stump: the mean cycle time measured 14 h, the TS-phase 6 h, the TG6 + M-phase 2 h and the TG1-phase 6 h. These data are discussed with respect to known cell cycle parameters of bladder urothelium regenerating in response to cytotoxic agents and of neoplastic urothelial cells. The reported findings provide a basis for further investigations using weak carcinogens and threshold doses of potent carcinogens to test the working hypothesis that stimulation of proliferation following partial cystectomy is capable of initiating, accelerating and/or potentiating carcinogenic cell transformation in the urinary bladder.

Double Labeling Autoradiography.Cell Kinetic Studies with 3H- and 14C-Thymidine 1.

Examples of the multiple applicability of the double labeling method with 3H- and 14C-TdR are demonstrated. Double labeling with 3H- and 14C-TdR makes it possible to determine the cycle and its phases with high precision by modifying the usual percent labeled mitoses method with a single injection of 3H-TdR. This results in a considerable improvement of the resolution of the percent labeled mitoses curve. In addition, data is provided on the variances of the transit times of the cells through the cycle phases. For example, in the case of the jejunal crypt cells of the mouse, the transit times through successive cycle phases are uncorrelated. In the case of glial cells the double labeling method provides cell kinetic parameters despite the paucity of proliferating glial cells. In the adult untreated animal, glial cell mitoses are so rare that the percent labeled mitoses method can not be utilized. However, the S-phase duration can be measured by double labeling and the cycle time can be determined by the so-called method of labeled S phases. With the latter method the passage through the next S phase of the 3H-TdR-labeled S phase cells can be registered by injecting 14C-TdR at different time intervals following 3H-TdR application. In this way an S-phase duration of about 10 hr and a cycle time of about 20 hr was found for glial cells in the adult untreated mouse. An exchange of glial cells between the growth fraction and the nongrowth fraction has also been shown by double labeling. A quite different application of the double labeling method with 3H- and 14C-TdR is the in vivo study of the cell cycle phase-specific effect of drugs used in chemotherapy of tumors. Up to now studies of the effect of cytotoxic drugs on cells in different cycle phases were confined to in vitro experiments, since such studies need synchronized cells. However, the double labeling method, which leads to well-defined subpopulations of differently labeled cells in different phases of the cycle, allows such studies to be carried out under in vivo conditions. The effect of vincristine on these cells has been studied. Vincristine affects cells in S and G2 in such a manner that they are arrested during the next metaphase and subsequently become necrotic. It has no effect on G1 cells.

Cell proliferation at different sites along the length of the rat colon.

This study has been undertaken in order to compare in detail cell proliferation in the mucosal crypts at several sites along the length of the large bowel of the rat. The techniques which have been used include simple morphometry, calculation of mitotic and tritiated thymidine labelling indices, metaphase arrest with vincristine, and the fraction of labelled mitoses method. Major differences exist in the size and shape of the crypts at different sites. In particular the distribution of the proliferating cells within the crypt varies. Mean cell cycle time ranges from 58 h in the descending colon to 25 h in the caecum; this variation appears to be brought about largely by changes in the duration of G1, the other phase durations remaining relatively constant. There is also variation in cell cycle time and growth fraction at different levels within the crypt; throughout the bowel cells appear to cycle more slowly at the bottom of the crypt, but changes in growth fraction do not display a similarly consistent pattern. Clearly the organisation of cell proliferation in the normal rat colon is very complex, and strict definition of anatomical location is required in any study of cell proliferation whether in normal or in diseased animals.

Temperature response of Catharanthus roseus cells cultivated in liquid medium

Suspension cultures of Catharanthus roseus G. Don cells were grown at various temperatures from 16°C to 40°C. The doubling time of cell populations was strongly increased by a fall in temperature from 27°C to 16°C. The cell cycle parameters of cell suspensions grown at 20°C and 27°C were analysed by the fraction of labelled mitoses method and compared. All cell cycle phases were affected by the shift in temperature with a higher sensitivity of the G 1 phase. Doubling times of cell populations and total cell cycle times were about the same at 20°C or 27°C. Consequently under these conditions all cells contributed to the growth of the populations. A strong influence of temperature on the amount per cell of the crude alkaloid fraction or on the specific content of serpentine and ajmalicine was observed. Lowering temperature from 27°C to 16°C resulted in a much higher alkaloid content, conversely when temperature was increased from 27°C to 38°C, the alkaloid content was strongly reduced.

Cell kinetic analysis of the quiescent cell compartment in cultured renal carcinoma of Syrian hamster.

Using a culture line of renal carcinoma of Syrian hamster (HKC-400), the distribution of DNA content in individual cells was analyzed by autoradiography after pulse and continuous labeling with 3H-thymidine in order to investigate the behavior of the non-proliferative cell compartment. Additional data used were the cell cycle parameters obtained by the fraction of labeled mitoses (FLM) method and the growth rate. The principle of analysis is to compare the cell fractions having G1 and G2 DNA contents with the fractions of G1 and G2 phase cells. It was concluded that even in the exponential phase of growth 31% of cells were blocked in either the G1 or G2 DNA region (Q2 = 22% and Q2 = 9%, and the cell loss rate from Q1 was higher than from Q2.