Distribution Of S-phase Cells Research Articles

The penny pusher: a cellular model of lens growth.

The mechanisms that regulate the number of cells in the lens and, therefore, its size and shape are unknown. We examined the dynamic relationship between proliferative behavior in the epithelial layer and macroscopic lens growth. The distribution of S-phase cells across the epithelium was visualized by confocal microscopy and cell populations were determined from orthographic projections of the lens surface. The number of S-phase cells in the mouse lens epithelium fell exponentially, to an asymptotic value of approximately 200 cells by 6 months. Mitosis became increasingly restricted to a 300-μm-wide swath of equatorial epithelium, the germinative zone (GZ), within which two peaks in labeling index were detected. Postnatally, the cell population increased to approximately 50,000 cells at 4 weeks of age. Thereafter, the number of cells declined, despite continued growth in lens dimensions. This apparently paradoxical observation was explained by a time-dependent increase in the surface area of cells at all locations. The cell biological measurements were incorporated into a physical model, the Penny Pusher. In this simple model, cells were considered to be of a single type, the proliferative behavior of which depended solely on latitude. Simulations using the Penny Pusher predicted the emergence of cell clones and were in good agreement with data obtained from earlier lineage-tracing studies. The Penny Pusher, a simple stochastic model, offers a useful conceptual framework for the investigation of lens growth mechanisms and provides a plausible alternative to growth models that postulate the existence of lens stem cells.

Immunogold-labeled S-phase neoblasts, total neoblast number, their distribution, and evidence for arrested neoblasts in Macrostomum lignano (Platyhelminthes, Rhabditophora)

Neoblasts in Platyhelminthes are the only cells to proliferate and differentiate into all cell types. In Macrostomum lignano, the incorporation of 5'-bromo-2'-deoxyuridine (BrdU) in neoblasts confirmed the distribution of S-phase cells in two lateral bands. BrdU labeling for light and for transmission electron microscopy (TEM) identified three populations of proliferating cells: somatic neoblasts located between the epidermis and gastrodermis (mesodermal neoblasts), neoblasts located within the gastrodermis (gastrodermal neoblasts), and gonadal S-phase cells. In adults, three stages of mesodermal neoblasts (2, 2-3, and 3) defined by their ultrastructure were found. Stage 1 neoblasts where only seen in hatchlings. These stages either were phases within the S-phase of one neoblast pool or were subsequent stages of differentiating neoblasts, each with its own cell cycle. Regular TEM and immunogold labeling provided the basis for calculating the total number of neoblasts and the ratio of labeled to non-labeled neoblasts. Somatic neoblasts represented 6.5% of the total number of cells. Of these, 27% were labeled in S-phase. Of this fraction, 33% were in stage 2, 46% in stage 2-3, and 21% in stage 3. Immunogold labeling substantiated results concerning the differentiation of neoblasts into somatic cells. Non-labeled stage 2 neoblasts were present, even after a 2-week BrdU exposure. Double labeling of mitoses and FMRF-amide revealed a close spatial relationship of mesodermal neoblasts with the nervous system. Immunogold-labeled sections showed that nearly 70% of S-phase cells were in direct contact or within 5 microm from nerve cords.

Proliferation of epithelia of noninvolved mucosa in patients with head and neck cancer.

Morphologically noninvolved mucosa in patients with head and neck cancer is altered by carcinogens. These alterations may include chromosome alterations, gene mutations, and other molecular abnormalities which may explain very high incidence of second tumors in this group of patients. The purpose of this study was to investigate the in vivo proliferative characteristics in epithelial tissues adjacent to the tumor in a series of patients with head and neck cancer. Twenty-one patients with head and neck tumors received IV infusions of iododeoxyuridine (IdUrd) and/or bromodeoxyuridine (BrdUrd). Surgical specimens containing normal-appearing epithelium adjacent to the tumor were selected and stained with the respective monoclonal antibody. The percentage of S-phase cells (labeling index, LI) was counted in the basal and suprabasal layers of the epithelium. In 27 samples of oral epithelium obtained from 14 previously untreated patients, labeled (S-phase) cells were predominantly located in suprabasal layers with LI 31.6 +/- 3.1% (range 13.5-73.2%). In contrast, the LI of the basal layer was very low: 1.6 +/- 0.2% (range 0.5%-8.8%). There was no statistically significant difference between normal appearing and dysplastic samples (p > 0.05). In 10 samples obtained from 7 patients whose biopsies were studied 2 days to 2 month after concomitant radiation and chemotherapy, the LI of the oral mucosa basal layer was significantly higher (21.0 +/- 4.1%, range 6.3-39.2%). The LI of the suprabasal layer in treated patients was 14.3 +/- 2.4% (range 5.9-31.1%). The LI of nasal pseudostratified epithelium (4 samples) was 11.2%. The average LI of "basal" cells was 8.3% (range 5.9-11.9%) and that of "suprabasal" cells was 13.8% range (3.2-29.5%). The basal layer of the skin (5 samples) contained 9.3% labeled cells (range 3.3-16.3%); the LI of suprabasal layers of skin was 21.3% (range 7.8-33.2%). Both the frequency and the spatial distribution of S-phase cells are disordered in noninvolved epithelia in patients with head and neck tumors. These observations suggest that disordered proliferation may be an early consequence of field cancerization, a consequence that occurs prior to appearance of morphologically apparent hyperplasia or dysplasia.

Proliferation of epithelia of noninvolved mucosa in patients with head and neck cancer

Background Morphologically noninvolved mucosa in patients with head and neck cancer is altered by carcinogens. These alterations may include chromosome alterations, gene mutations, and other molecular abnormalities which may explain very high incidence of second tumors in this group of patients. The purpose of this study was to investigate the in vivo proliferative characteristics in epithelial tissues adjacent to the tumor in a series of patients with head and neck cancer. Methods Twenty-one patients with head and neck tumors received IV infusions of iododeoxyuridine (IdUrd) and/or bromodeoxyuridine (BrdUrd). Surgical specimens containing normal-appearing epithelium adjacent to the tumor were selected and stained with the respective monoclonal antibody. The percentage of S-phase cells (labeling index, LI) was counted in the basal and suprabasal layers of the epithelium. Results In 27 samples of oral epithelium obtained from 14 previously untreated patients, labeled (S-phase) cells were predominantly located in suprabasal layers with LI 31.6 ± 3.1% (range 13.5–73.2%). In contrast, the LI of the basal layer was very low: 1.6 ± 0.2% (range 0.5%–8.8%). There was no statistically significant difference between normal appearing and dysplastic samples (p > 0.05). In 10 samples obtained from 7 patients whose biopsies were studied 2 days to 2 month after concomitant radiation and chemotherapy, the LI of the oral mucosa basal layer was significantly higher (21.0 ± 4.1%, range 6.3–39.2%). The LI of the suprabasal layer in treated patients was 14.3 ± 2.4% (range 5.9–31.1%). The LI of nasal pseudostratified epithelium (4 samples) was 11.2%. The average LI of “basal” cells was 8.3% (range 5.9–11.9%) and that of “suprabasal” cells was 13.8% range (3.2–29.5%). The basal layer of the skin (5 samples) contained 9.3% labeled cells (range 3.3–16.3%); the LI of suprabasal layers of skin was 21.3% (range 7.8–33.2%). Conclusion Both the frequency and the spatial distribution of S-phase cells are disordered in noninvolved epithelia in patients with head and neck tumors. These observations suggest that disordered proliferation may be an early consequence of field cancerization, a consequence that occurs prior to appearance of morphologically apparent hyperplasia or dysplasia. HEAD & NECK 1996;18:522–528 © 1996 John Wiley & Sons, Inc.

Cell kinetics in rat small intestine after exposure to 3 Gy of gamma-rays at different times of the day.

Qualitative and quantitative morphological changes in rat jejunum were studied after a whole-body exposure to 3 Gy of gamma rays. Four groups of animals were irradiated at different times of the day, namely midnight, 06.00, 12.00 and 18.00 hours. The number of epithelial cells, labelling and mitotic indices were evaluated in crypt sections and the spatial distribution of S-phase cells was determined. At 12 h after irradiation a marked reduction was observed in all parameters, but the proliferative activity was restored quickly and at 36 h after irradiation the values were significantly higher than the controls. The frequency distribution of labelled cells at different positions in the crypt was reduced at 12 h but a clear expansion of S phase cells to positions near to the crypt villus junction was observed during the recovery phase. The animals irradiated at different times of the day showed a similar general post-irradiation response in the number of cells along the side of the crypt, labelling and mitotic indices and in the distribution of S phase cells along the crypts. It is worth noting that the animals exposed at midnight had a distribution of S phase cells similar to controls at 72 h post-irradiation, i.e. earlier than the other groups.

Diagnosis of gastric adenoma versus early gastric cancer by bromodeoxyuridine immunohistochemistry from gastric biopsy specimen.

Differentiation between gastric adenomas and type IIa early gastric cancer by macroscopic and histologic examinations is often difficult. This study has shown that there are differences in the distribution of S-phase cells in these two types of tumour, and these differences can be exploited to differentiate between them. The distribution of S-phase cells was assessed by Bromodeoxyuridine (BrdU) immunohistochemistry using an in vitro BrdU-labelling method. Biopsy specimens were obtained from 27 lesions (12 adenomas and 15 type IIa early gastric cancers), and the distributions of BrdU-labelled cells in the tumour thickness as well as in the superficial layer of the tumour were determined. Significant difference was seen in the labelling of the two tumour types with regard to both these parameters. Using the criteria of BrdU immunohistochemistry, the accuracy of differentiation between gastric adenomas and cancers was 88.9%. Therefore, it is concluded in this study that in vitro BrdU labelling is a useful method of diagnosing gastric adenomas and cancers.

Hydrocortisone perturbs the cell proliferation pattern during feather morphogenesis: evidence for disturbance of cephalocaudal orientation.

In this study, we have monitored the spatial distribution of S-phase cells during successive stages of normal feather morphogenesis using the specific marker BrdU. We also disturbed the development program by administration of hydrocortisone on the chorioallantoic membrane of 6.5-day chick embryos and examined the resulting pattern of BrdU incorporation. Our results show that a specific spatio-temporal pattern of cell proliferation occurs during successive stages of feather development and that this pattern accounts for the growth of feather buds according to the cephalocaudal orientation. Our experimental analysis showed that the stage-dependent alteration of feather morphogenesis (as shown by Züst, Ann. Embryol. Morphogen. 4, 1971 and confirmed by Démarchez et al., Dev. Biol. 106, 1984), is based on a stage-dependent alteration of the proliferation pattern in the epidermis. Forty-eight hours after treatment, non-induced epidermis ceases DNA synthesis and is unable to form placodes. Induced epidermis at the placodal and dermal condensation stages fails to produce the cohorts of S-phase cells responsible for the caudal outgrowth and the slanting shape of the buds. These young buds display anarchic proliferation in the whole epidermis possibly resulting in the appearance of "curly" feathers. Together, these results show the importance of the spatial pattern of ectodermal and mesodermal cell proliferation during the normal feather morphogenesis. Moreover, they corroborate the particular role of epidermis both in the establishment of feather rudiments and in the cephalocaudal orientation of the feathers.

The relationship between cell proliferation and the transcription of the nuclear oncogenes c-myc, c-myb and c-ets-1 during feather morphogenesis in the chick embryo

We have described the expression of three nuclear protooncogenes, c-myc, c-myb and c-ets-1 during feather morphogenesis in the chick embryo. In parallel with the expression patterns obtained by in situ hybridization, we have mapped the spatial distribution of S-phase cells by monitoring the incorporation of 5-bromodeoxyuridine. We do not detect c-myc or c-myb transcripts during the early stages when S-phase cells are scattered in the dermis and in the epidermis. Rather c-ets-1 transcripts are abundant in the dermal cells which divide and accumulate under the uniform epidermis. At the onset of the formation of the feather bud, cells within each rudiment cease DNA replicative activities and c-myc transcripts are detected both in the epidermis and in the underlying dermis. This expression precedes the reentry into the S phase. The transcription of c-myb, which has been previously tightly linked to hemopoietic cells is also detected in the developing skin. This expression is essentially located in proliferating epidermal cells on and after the beginning of feather outgrowth. As feather outgrowth proceeds, the distribution of c-myc and c-myb transcripts is restricted to the highly proliferating epidermis. In contrast c-ets-1 transcripts are never detected in the epidermis. During the later stages of skin morphogenesis, the transcription of c-ets-1 is restricted to the endothelial cells of blood vessels, as previously described. We suggest that the differential expression of these nuclear oncogenes reflects the activation of different mitotic controlling pathways during the development of the skin.

Does response and proliferative characteristics of aberrant crypt foci: putative preneoplastic lesions in rat colon

Foci of aberrant crypts (ACF) have been identified in the unsectioned methylene blue stained rodent colons and hypothesized to represent precursor lesions of colon cancer. In the present study, induction and growth characteristics of ACF were investigated in response to a single injection of varying dosages of 1,2-dimethylhydrazine-2HCl (DMH), a colon carcinogen. Female Sprague-Dawley rats were given a single injection of DMH (5-150 mg/kg). Two and 19 weeks after the injection, animals were killed and their distal 10 cm of colons were enumerated for the number and crypt multiplicity of ACF. Number of ACF increased with increasing dosages of DMH plateauing at 100 mg/kg. However, percentage of ACF exhibiting different crypt multiplicity (1 to greater than 4) were similar among different dose groups. Aberrant crypts and normal crypts were enumerated for total number of cells and number and distribution of S-phase cells along the crypt height 19 weeks after DMH injection after autoradiography. The labeling index (LI) (percentage of S-phase cells) and LI along the crypt height were determined. Compared to the surrounding normal crypts, aberrant crypts exhibited significantly higher (P less than 0.05) number of cells (1122 +/- 81 versus 411 +/- 28) and higher (P less than 0.05) LI (21 +/- 1 versus 12 +/- 1). For the eight ACF analysed in the present study, the distribution of S-phase cells in the aberrant crypts were similar to that of normal crypts in that S-phase cells were restricted to the lower two-thirds of the crypts rather than distributed throughout the height of the crypts as reported for adenomatous epithelium.

Application of Bromodeoxyuridine Immunohistochemistry to the Rat Temporomandibular Joint

To detect S-phase cells in the decalcified paraffin-embedded temporomandibular joint (TMJ) tissues of the rat, bromodeoxyuridine (BrdUrd) immunohistochemistry was performed. Percardiac perfusion of 10% EDTA-2Na Tris buffer accelerated the decalcification. The use of the monoclonal antibody to BrdUrd enabled the visualization of the distribution of S-phase cells. This method is practical in studying cell kinetics and the specific proliferation mode of the TMJ.

The effect of dietary omega-3 fatty acids (fish oil) on azoxymethanol-induced focal areas of dysplasia and colon tumor incidence

MaxEPA (MA), a fish oil high in omega-3 fatty acids, was combined with various levels of corn oil (CO), rich in omega-6 fatty acids, and fed to female CF1 mice. The three fish oil blends with CO and the two CO levels of the diets studied were as follows: 16.0% CO + 4.4% MA (Diet 1); 10.2% CO + 10.2% MA (Diet 2); 4.4% CO + 16.0% MA (Diet 3); 20.4% CO (Diet 4); and 4.4% CO (Diet 5). The diets were provided 2 weeks before weekly subcutaneous injection of saline or azoxymethanol (AOM). Studies of epithelial cell proliferation and the incidence of focal areas of dysplasia (FAD) involved six weekly AOM injections. One week after the last AOM injection and 1 hour before killing, mice were injected with tritiated thymidine (3HTdR). No differences in any proliferative parameters were found among the five groups of saline-treated mice. Among the AOM-treated animals, those fed Diet 3 showed significantly fewer cells per crypt and significantly fewer labeled cells/gland than CO Diets 4 and 5. Additionally, the distribution of S-phase cells in crypts of AOM-treated mice fed Diet 3 most closely resembled that of the saline controls. The greatest alteration in the distribution of proliferative cells was observed in the high-CO diet (Diet 4) and the lowest MA level (Diet 1). Mice fed Diets 2 and 3 had significantly fewer FAD/500 microns of distal colonic serial sections than those fed the high CO diet (Diet 4). Mice involved in chronic tumor incidence studies received only three weekly injections of the same dose of AOM. Regardless of diet, approximately 88% of all tumors arose in the distal colon. A significantly larger tumor-bearing population was observed in both the high-CO Diet 4 and the lowest MaxEPA (MA) diet (Diet 1) compared with the incidence in MA Diets 2 and 3 and the low-CO Diet 5. A diet with a ratio of omega-6 to omega-3 fatty acids of approximately 1.0 apparently prevented the development of an adenoma-type proliferative pattern thereby reducing FAD numbers and subsequent tumor incidence.

Late cytokinetic abnormalities in irradiated reactal mucosa

Late cytokinetic changes of the colonic crypt epithelium after radiation therapy were investigated. A monoclonal antibody to bromodeoxyuridine (anti-BrdU MAb) was used in tissue specimens previously incubated with BrdU to show S-phase cells by immunohistochemical technique. Endoscopic rectal biopsies were taken from 30 patients previously treated with radiotherapy for gynaecological cancer and from 50 patients with comparable but untreated neoplasms, as controls. Number and height distribution of S-phase cells were evaluated by dividing each crypt column into 5 equal longitudinal compartments. No statistically significant differences were found in total Labelling Index (LI) between controls and irradiated mucosa, whereas LI per crypt compartment, percentage of labelled compartments and percentage of BrdU-positive cells in the middle and superficial portions of the crypt were significantly higher in patients submitted to radiation therapy. This kinetic abnormality corresponds to a progressive shift of the major zone of DNA synthesis to the upper third of the crypt as a late reaction to radiation and represents an early step in the histogenesis of colorectal cancer. These results lend support to the view that there is a higher risk of colorectal carcinoma after pelvic irradiation.

Bromodeoxyuridine 標識法により観察したラット膀胱前癌病変におけるS期細胞の割合及び分布状態の変化

We studied whether bromodeoxyuridine (BrdU)-labeling method showing the population and/or distribution of S-phase cells in tumor tissues is useful to detect the precancerous lesions of bladder. The bladder lesions were induced by 0.05% N-butyl-N (4-hydroxybutyl) nitrosamine (BBN) in drinking water for ten weeks in five male rats (Wistar-Imamichi). BrdU (50 mg/kg) was injected to intraperitoneal space two hours before the resection of the bladder. The detection of positive cells (S-phase cells taking BrdU) was performed by peroxidase-antiperoxidase method. From five bladder specimens 144 lesions (simple hyperplasia (S)-73, papillary or nodular hyperplasia (PN)-57, papilloma (Pa)-14) were analysed. The labeling index (the ratio of positive cells to all epithelial cells in one lesion) showed no significant difference among the lesions (S, PN, and Pa). For analyses of the distribution patterns of S-phase cells, we investigated whether positive cells aside from basal layers and/or the serial positive cells in basal layers were seen or not in each lesion. The ratios of lesions containing positive cells aside from basal layers were 11% (8/73), 60% (34/57) and 100% (14/14) in S, PN and Pa lesions. The serial positive cells in basal layers were observed in 6% (4/73), 25% (14/57) and 50% (7/14) of S, PN and Pa lesions. Those differences were statistically significant. Above mentioned results suggest that the appearance of S-phase cells aside from basal layers and/or the serial S-phase cells in basal layers is characteristic change in precancerous lesions, which is useful for sooner detection of bladder carcinogenesis.

DNA synthesis, blood flow, and glucose utilization in experimental rat brain tumors

The relationships between distribution of deoxyribonucleic acid (DNA)-synthesizing cells (S-phase cells) and blood flow and glucose utilization were investigated in rat brain tumors using an autoradiographic technique and immunoperoxidase staining for bromodeoxyuridine (BUdR). Two strains of rat brain tumor were used: strain A and B, both induced by the Rous sarcoma virus. Strain A was biologically more malignant than strain B. The blood flow was unevenly distributed in the tumor, compared with the contralateral cortex, the average blood flow in the tumor was about 50% in strain A and 60% in strain B. The distribution of blood flow did not correlate with the distribution of S-phase cells or with the distribution of vessels in the tumor in either strain A or B. The average glucose utilization in strain A was about 250% and in strain B about 170% of that of the contralateral cortex. The high glucose utilization area correlated well with the distribution of BUdR-positive nuclei in strain B. These findings suggest that the biological malignancy of a tumor correlates with glucose utilization rather than with blood flow, and that malignant brain tumors show a marked increase in glucose utilization for nucleic acid synthesis.

Rectal epithelial cell proliferation in a group of young adults. Influence of age and genetic risk for colon cancer.

Twenty-one medical students with an average age of 23.3 +/- 1.5 years and no family history of large bowel cancer had a rectal biopsy taken for in vitro incorporation of tritiated thymidine (3HTdR). The number and position of labeled epithelial cells in this group was compared with an older control group of 12 and two groups of individuals at high risk for colon cancer. The latter were (1) 18 young asymptomatic individuals from familial colon cancer families without polyposis with a 50% risk for colon cancer, average age 24.1 +/- 3.7; and (2) 17 older individuals with the same genetic background and risk factor, average age 53.8 +/- 10.2 years. A group of 20 young individuals from cancer-free branches of familial colon cancer kindreds with a 25% risk for colon cancer, average age 23.1 +/- 3.6, also was studied. No overall differences in labeling index (LI) were found among the young groups, i.e., the control versus the low risk or high risk. The range of LI values was narrow regardless of risk. However age was associated with a significant increase in LI as observed among older controls and the 50% risk group as compared to younger persons; the range of LI values was wider among the older groups regardless of risk. Concerning the distribution of DNA-synthesizing cells, young controls had significantly more S-phase cells in the lower third of the crypts than either young high-risk or low-risk groups (P less than 0.05). Similarly, young controls had significantly fewer labeled cells in the middle third than the young high-risk group and young controls had significantly fewer S-phase cells in the upper third than did young low-risk or high-risk groups. When young and old members of the same group were pooled, the control group had the highest percentage of labeled cells in the lower third of the crypt (62.5%) which was significantly higher than both the low-risk (53.7%) and the pooled high-risk groups (51.6%). For the upper third of the glands, the low-risk (7.6%) and the pooled high-risk groups (6.8%) were significantly different from the controls (3.3%). Thus initial risk for colon cancer is more related to the distribution of S-phase cells than to their number, i.e., LI. Risk appears to be related to more DNA-synthesizing cells in the upper crypt, an indication that relatively early on in life the DNA switch-off mechanism shows signs of being defective.

Cell proliferation and colonic neoplasia.

The preneoplastic events occurring in the mucosa of patients at risk of developing colonic cancer are described and correlate well with the histogenesis of an adenoma. Confirmatory evidence from experimental carcinogenesis systems is provided. Among individuals at 50% risk of colon cancer, proliferative abnormalities relating to the distribution of S-phase cells are present. Aging, whether among the high-risk or the unaffected control population, appears to bring about enhancement of the percentage of DNA-synthesizing cells in the crypts, indicating a similar response of colonic epithelial cells to environmental conditions over time. The combination of an already existing defect in cell proliferation among those at high risk of familial colon cancer along with an elevated rate of cell renewal increases the probability that a neoplasm will arise in this population.

Distribution of S-phase cells during the regeneration of Drosophila imaginal wing discs

We investigated the distribution of S-phase cells during regeneration of the imaginal wing disc of Drosophila melanogaster following excision of 30°, 90°, and 150° sectors of tissue. The fragments were cultured in adult abdomens for 1–5 days, labeled in vitro with tritiated thymidine, serially sectioned, and subjected to autoradiography. There was negligible thymidine incorporation in unoperated controls and in the undamaged parts of the operated discs, indicating that DNA synthesis in undamaged tissue is terminated during the first day of the culture period. Almost all of the fragments from which tissue had been removed, as well as controls which were simply cut without the removal of any tissue, showed a cluster of labeled cells (blastema) even after only 1 day of culture. The blastemas in control discs were short-lived, with over 50% of these discs showing no blastema by the third day in culture. Blastemas in discs from which sectors were removed were more persistent; the time at which 50% of the fragments no longer showed a blastema was 4 days for the −30° fragments, 5 days for the −90° fragments, and greater than 5 days for the −150° fragments. The average blastema size, measured as number of labeled cells, was directly related to the amount of tissue removed, and in most cases did not change significantly during the culture period. Both wound edges incorporated tritiated thymidine initially and the S-phase cells remained tightly clustered throughout regeneration; maximum blastema width varied from about 8 to 25 cell diameters. The results are consistent with the idea that regenerative cell proliferation is stimulated and maintained by positional information discontinuities, and terminated when these discontinuities are resolved by the addition of an appropriate number of new cells.

DNA-Measurements on Formalin-fixed, Paraffin-embedded Squamous Cell Carcinomas from different ENT-Regions

DNA measurements were performed on formalin-fixed, paraffin-embedded squamous cell carcinomas, using the method of Hedley et al. Ultrasonication was sometimes necessary after enzymatic disintegration to dispose of residual cytoplasm. The isolated nuclei were stained with Hoechst 33258. DNA measurements were performed using a rapid system for static fluorometry (FLUORA-programme). The method proved to be highly accurate. The mean CV of the stem cell peaks from different tumours was 5.3. There was no difference between material less or more than 5 years old (mean CVs 5.2 and 5.5 respectively). RNase treatment had no effect and was therefore not considered necessary. The proliferative activity was calculated by computer assuming a rectilinear distribution of S-phase cells. Because routinely paraffin-embedded material can be used, DNA analysis can be performed on tumour material where the clinical outcome is known, in order to evaluate certain DNA variables.

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.

Modifications of S-phase cell distribution in the intestinal crypts after multiple daily fractionation.

The effects obtained by multiple daily fractionation (3 Gy X 2 or 3 Gy X 2 X 2) on the distribution of S-phase cells along the crypt of the small intestine were investigated. The frequency of labelled cell distribution was reduced at early intervals; then the proliferating compartment gradually extended to the villus junctions. During recovery labelled cell frequency in the lower half of the crypts returned to control levels, while labelled cells were present in the differentiating area. With lower total dose modifications were milder and, as early as 72 h before exposure, distribution was already similar to controls. Invertase activity showed an initial increase and a higher reduction during acute damage when fractionation with higher doses was used. A lack of return to normal activity was present even 11 days after exposure when, however, the characteristic circadian pattern was observed.