Decrease In Labeling Index Research Articles

Effects of Femara and Tamoxifen on Proliferation of FM3A Cells in Culture

In this study, antiproliferative effects of the selective estrogen receptor modulator Tamoxifen and the aromatase inhibitor letrozole (Femara) were evaluated and compared using the FM3A cell line, originating from a C3H mouse mammary carcinoma and positive in terms of estrogen receptor (ER) expression. Cell kinetic parameters including labelling index, mitotic index and labelling index were assessed after exposure of the. FM3A cell line to 0.001μg/ml of Tamoxifen and 0.25μg/ml of Femara for 4, 8, 16 and 32 h for all parameters. The results showed that cell growth was inhibited by both agents. There was a significant decrease in labelling index and mitotic index and significant increase in apoptotic index for all experimental groups. The differences between control and all experimental groups were statistically significant (p<0.001) for all applications.

Dietary fiber decreases colonic epithelial cell proliferation and protein synthetic rates in human subjects

Although it has been proposed that high fiber consumption can prevent proliferative diseases of the colon, the clinical data to support this hypothesis have been inconsistent. To provide a more robust measure of the effects of fiber on colonic mucosal growth than previous studies, we evaluated both cell proliferation and colonic mucosal protein synthesis in nine healthy volunteers after they consumed a typical Western diet (<20 g fiber/day) or a Western diet supplemented with wheat bran (24 g/day) in a randomized crossover design. Biopsies taken from the sigmoid colon were used to assess mucosal proliferation by determining proliferating cell nuclear antigen (PCNA) in crypt cells and to assess mucosal protein synthetic rate using stable isotopically labeled leucine infusion. Fiber supplementation produced a 12% decrease in labeling index (%crypt cells stained with PCNA) (P < 0.001) and an 11% decrease in mucosal protein fractional synthetic rate (FSR; P < 0.05). Moreover, mucosal protein FSR correlated directly with labeling index (r2= 0.22, P < 0.05). These data demonstrate that increased wheat bran consumption decreases colonic mucosal proliferation and support the potential importance of dietary fiber in preventing proliferative diseases of the colon.

Effects of Relatively Low Levels of Mono-(2-Ethylhexyl) Phthalate on Cocultured Sertoli Cells and Gonocytes from Neonatal Rats

Di-(2-ethylhexyl) phthalate (DEHP), one of the abundant man-made environmental chemicals, induces testicular damage in both developing and adult animals. However, the nature and mechanism underlying the action of phthalates on testicular development remain largely unexplored. In the present study, we used cocultures of neonatal Sertoli cells and gonocytes (precursors of spermatogonia) to characterize in detail the effects of mono-(2-ethylhexyl) phthalate (MEHP; the active metabolite of DEHP) on these cells and to explore the underlying mechanism(s). Sertoli cells and gonocytes were isolated from rat pups on the 2nd day after birth, cocultured, and exposed to MEHP at concentrations of 0.01, 0.1, or 1.0 μM, or to 0.5% DMSO (vehicle control), or 10 μM DEHP (negative control) for a total of 48 h. We found that exposure to MEHP induced gonocyte detachment from the Sertoli cell monolayers in a time- and dose-dependent manner. When exposed to 1.0 μM MEHP, many gonocytes started to detach after 12 h of exposure and most gonocytes were lost during the media change at 24 h. Gonocyte detachment was also observed in cocultures treated with 0.1 μM MEHP for 24 h of exposure, but not in cultures treated with 0.01 μM MEHP for 48 h. Detached gonocytes were viable as indicated by their ability to exclude trypan blue. Furthermore, when proliferation of cultured Sertoli cells was detected by BrdU labeling and subsequently quantified, we found that exposure to 0.1 or 1.0 μM MEHP for 48 h resulted in a decrease in labeling indices of 33.6 and 83.6%, respectively, compared to the vehicle control (p< 0.01), while the labeling index was unchanged by treatment with 0.01 μM MEHP. In addition, we also tested the potential effect of MEHP on FSH-stimulated Sertoli cell proliferation by simultaneously treating cultures with 200 ng/ml human FSH and different concentrations of MEHP for 48 h. Exposure to 0.1 or 1.0 μM MEHP resulted in decreases of 24.2 and 74.2%, respectively, in FSH-stimulated Sertoli cell proliferation (p< 0.01). Furthermore, MEHP also inhibited dibutyl cAMP-stimulated Sertoli cell proliferation, regardless of whether dibutyl cAMP was added to the cultures before or at the same time as MEHP. Finally, addition of FSH or dibutyl cAMP had no effect on MEHP-induced gonocyte detachment, and none of the observed effects on either Sertoli cells or gonocytes were detected in control cultures treated with 0.5% DMSO only or with 10 μM DEHP. Therefore, short exposure to low levels of MEHP disrupted adhesion of gonocytes to Sertoli cells and inhibited both basal and FSH-stimulated Sertoli cell proliferation in a dose-dependent manner. The lowest effective dose of MEHPin vitrowas 0.1 μM, which is about 10- to 1,000-fold lower than the dose shown to affect Sertoli cells from prepubertal animals. Moreover, our data indicate that MEHP impairs division of neonatal Sertoli cells by acting at a post-cAMP site in the FSH-response pathway or via a mechanism independent of FSH. These data provide direct new evidence that relatively low levels of MEHP disrupt Sertoli cell–gonocyte physical interactions and suppress Sertoli cell proliferation in neonates via mechanisms specific to neonatal testis where the foundations of adult fertility are established. The results also highlight the neonatal period of testicular development as one particularly sensitive to environmental chemicals.

Short- and long-term oestradiol treatment inhibits growth and alters expression of p21WAF1/Cip1 in an endometrial adenocarcinoma cell line lacking functional p53.

Oestrogen is assumed to play a significant role in cell cycle regulation of cells expressing the oestrogen receptor, although its mechanism of action is not yet well defined. To examine this, a mutant p53-expressing human endometrial adenocarcinoma cell line of the oestradiol-inhibited growth phenotype was treated with oestradiol for 2 weeks (short-term) and 6 months (long-term). With short-term treatment, cells were treated with increasing doses of oestradiol. The highest dose, 1 microM, was used in the long-term interval. The influence of the hormone on growth, proliferation and expression of some cell cycle and apoptosis-related proteins was evaluated. In cells treated for 2 weeks, there was a dose-dependent inhibition of both growth and proliferation with a significant decrease in labelling index (LI) and S-phase fraction (SPF) and a simultaneous increase in the fraction of cells in G0/G1. Extending the oestradiol treatment to 6 months showed further growth retardation and decreased proliferation with cells accumulating in G0/G1. Analysis of the expression of p21WAF1/Cip1 showed a nearly 2-fold increase after 2 weeks treatment with 1 microM oestradiol, which was also observed after long-term treatment without any further increase in protein levels. Expression of the anti-apoptotic protein bcl-2 was not affected after short-term treatment but decreased significantly after 6 months treatment compared to control cells. Our results suggest the existence of a p53-independent pathway of oestradiol regulation of growth and proliferation in this human endometrial adenocarcinoma, resulting in accumulation of cells in G0/G1 through p21WAF1/Cip1 induction and, after prolonged treatment, downregulation of bcl-2 protein.

Decreased muscle cell proliferation in chicks with a deletion in the GH receptor gene

The increase in muscle weight in neonatal animals is a consequence of increased protein accretion and DNA content. GH increases protein accretion but direct effects of GH on myogenic cell proliferation have not been demonstrated. Sex-linked dwarfism in the chick is caused by mutation or deletion in the GH receptor gene and has provided a useful model to study the physiological consequences of GH insensitivity. This study determined the consequences of GH receptor gene mutation on muscle cell proliferation in vivo. Northern and Southern blotting and PCR analysis revealed restriction fragment length polymorphism patterns and a 1.7 kb deletion of the intracellular domain of the GH receptor gene in commercial dwarf broiler chicks, similar to the Connecticut strain in which there is a dysfunctional GH receptor. Cell proliferation was measured in muscle sections from normal and dwarf chicks after incorporation of 5-bromo-2'-deoxyuridine (BrdU; 25 mg/kg) in vivo at 2, 5 and 13 days of age. Incorporation of BrdU into nuclei was measured in frozen sections, counterstained with propidium iodide to estimate the total number of nuclei by quantitative image analysis, and the labelling index was calculated. Paraffin-embedded sections of breast muscle were stained using an anti-human IGF-I polyclonal antibody. Expression of IGF-I mRNA in muscle from each genotype at 5 days of age was measured by RNAse protection assay. The labelling index was similar in 2-day-old chicks from both genotypes (normal, 20.14 +/- 2.39%; dwarf, 19.79 +/- 5.83%). By day 5 the labelling index had decreased but was significantly higher (P < 0.02) in normal (12.53 +/- 3.36%) compared with the dwarf (6.25 +/- 1.39%). By 13 days of age, there was a further decrease in labelling index but no difference between the groups (normal, 4.92 +/- 1.28%; dwarf, 4.96 +/- 1.51%). IGF-I mRNA was expressed and IGF-I peptide was identified in muscle sections but there was no difference between genotypes. The results show that cell division in breast muscle in vivo is high in neonatal chicks but it declines with increasing age. The absence of a functional GH receptor in the dwarf is associated with a greater decline in DNA synthesis and suggests that GH may directly affect a proportion of cells, since there was no difference in IGF-I mRNA or peptide.

Sulindac and polyp regression.

Sulindac is useful in regression of adenomatous polyps. In addition to orally administered sulindac, rectal preparations also appear to be efficacious [32]. However, further studies are necessary to determine whether regression of adenomas, the precursor of colorectal cancer, will cause decrease in colorectal cancer risk in both FAP and non FAP patients. Moreover, clinical studies are needed to test the application of this potential chemopreventative drug in several other patient populations. Several interesting observations have been made concerning the use of sulindac. Indomethacin, a related NSAID to sulindac, did not cause polyp regression. Also, upper gastrointestinal tract polyps in the stomach and duodenum appear not to be affected by sulindac therapy. These observations might be explained by the metabolism of sulindac in which the pharmacologically active sulfide metabolite is generated and distributed in the large intestine. Also, investigators noted that after discontinuation of sulindac adenomas recurred. Sulindac treatment was well tolerated at the usual clinical doses. Although some investigators have speculated on the effect of prostaglandin inhibition sulindac on cAMP-dependent mechanisms which control cellular proliferation [33], the cause of adenoma regression is unknown. There is evidence that colorectal endogenous prostaglandin levels decrease with sulindac. Evaluation of colorectal mucosal cellular proliferation in patients treated with sulindac has revealed a decrease in labelling index by bromodeoxyuridine labeling in one study [28] but no change in labelling index by [3H]thymidine incorporation in another investigation [34]. Also, ki 67 labelling index, another measure of cellular proliferation, was not affected in the colorectal mucosa of patients taking sulindac.(ABSTRACT TRUNCATED AT 250 WORDS)

Octreotide (SMS 201–995) inhibits the growth of colon peritoneal carcinomatosis in BDIX rats

In order to assess the effect of octreotide, a somatostatin analogue, on the growth of colon peritoneal carcinomatosis, 20 BDIX rats were injected i.p. with 1·10 6 colon cancer cells (DHD/K12 tumor cell line) and received octreotide, 65 μg/kg s.c. every 12 h ( n=10) or saline ( n=10) for 42 days, starting 3 days after tumor cell injection. Animals were killed at the end of the treatment. The mean volume of ascites was lower in the octreotide group ( 33.7±7.6 ml ), than in the control group ( 67.5±16.3 ml ; P<0.05). The extent of peritoneal carcinomatosis (in five classes according to a previously published classification) was lower in the octreotide group ( P<0.05). Cell proliferation, using the BrdU technique, was markedly inhibited by octreotide (labeling index of tumor cells: 17.0±0.6% vs. 26.3±2.2% in controls, P<0.001). No significant decrease in labeling index was observed in normal colonic mucosa. Two subtypes of somatostatin receptors were found in all tumors, using the 30F3 monoclonal antireceptor antibody. K D and B max values were not significantly different in the octreotide and control groups: high affinity, low capacity receptors ( K D =1.4·10 −10 M and 0.7·10 −10 M , respectively; B max =3.8 and 2.9 pmol/mg protein, respectively); low affinity, high capacity receptors ( K D =1±0.2·10 −9 M , respectively; B max =27.8±0.1 and 22.8±0.05 pmol/mg protein, respectively). Octreotide inhibits the growth of colon peritoneal carcinomatosis in vivo in BDIX rats through an inhibition of DNA synthesis; this effect is at least partly mediated by somatostatin receptors present on tumor cells.

Effect of magnesium hydroxide on methylazoxymethanol acetate-induced epithelial proliferation in the large bowels of rats

The effect of magnesium hydroxide on the epithelial proliferation of the large bowel was examined using rats given methylazoxymethanol (MAM) acetate. Dietary administration of magnesium hydroxide at 250, 500, 1000 or 2000 ppm. for 1, 3 or 5 weeks did not influence the cell cycle of the cryptal cells of the large bowel. However, the exposure to magnesium hydroxide under these conditions lowered the bromodeoxyuridine labeling index of the cells of the large bowel of the rats which had been initiated by MAM acetate (25 mg/kg, 3 times). The decrease in labeling index was more apparent in the proximal segment than in the distal segment. Such an inhibitory effect on the DNA synthesis of the epithelial cells by magnesium hydroxide may be related to the suppressive action of the trace element on the carcinogen-induced large bowel carcinogenesis.

The effect of epidermal growth factor on neonatal incisor differentiation in the mouse

The effect of epidermal growth factor (EGF) on cellular differentiation of the neonatal mouse mandibular incisor was examined autoradiographically using tritiated thymidine ([ 3H]TDR) and tritiated proline ([ 3H]PRO). On Days 0 (day of birth), 1, and 2, EGF was administered (3 μg/g body wt) sc to neonates. Mice were killed on Days 1, 4, 7, 10, and 13 after birth and were injected with either [ 3H]TDR or [ 3H]PRO 1 hr before death. [ 3H]TDR was used to analyze cell proliferation in eight cell types in the developing mouse incisor including upper (lingual) and lower (buccal) pulpal fibroblasts, preodontoblasts, inner and outer enamel epithelial cells (IEE and OEE), stratum intermedium (SI), stellate reticulum (SR), and periodontal ligament (PDL) fibroblasts. [ 3H]PRO was used to analyze protein synthesis in ameloblasts, and their secretion products (enamel and dentin), as well as PDL fibroblasts. The selected EGF injection scheme elicited acceleration of incisor eruption with minimal growth retardation. At Day 1, the upper and lower pulp, preodontoblasts, SI, and SR showed a significant decrease in labeling index (LI) 24 hr after a single EGF injection. After multiple injections (Days 0, 1, 2), two LI patterns were observed. In lower pulp, preodontoblasts, IEE, SI, SR, and OEE, a posteruptive change in LI was observed. In contrast, the upper pulp and PDL regions demonstrated a direct temporal relationship with eruption. Autoradiographic analysis with [ 3H]PRO indicated that EGF treatment caused significant increases in grain counts per unit area in ameloblast, odontoblast, and PDL regions studied. Significant differences were found in all four regions studied (ameloblasts, enamel, odontoblasts, dentin) at the 45-μm-tall ameloblast level as well as ameloblasts and odontoblasts at the 30-μm level at 13 days of age. The PDL demonstrated significant differences at all locations studied (base, 30 μm, 45 μm,) in 4-, 7-, and 13-day-old mice. Morphologically, EGF-treated groups demonstrated premature differentiation of ameloblasts and odontoblasts at the light microscopic level. The data indicate that EGF alters DNA and protein synthesis as well as differentiation patterns during the eruption process. While EGF affects both DNA and protein synthesis, the alteration of differentiation may be secondary to mitogenic effects on proliferative compartments. In order to determine the cellular target for EGF within the newborn mouse incisor, in vivo 125I-EGF binding was analyzed autoradiographically. EGF binding sites were demonstrated in the dental follicle, an area which contains cells believed to be the progenitors for the PDL, alveolar bone, and cementum. The results of the [ 3H]TDR, [ 3H]PRO, and 125I-EGF studies argue strongly that the PDL is the target for EGF within the incisor.

Developmental behavior of N,N'-dimethylnitrosourea-induced brain gliomas and influence of a stab wound in adult rats.

The developmental behavior of rat brain gliomas induced by weekly injections of N,N'-dimethylnitrosourea (DMNU) from the age of three weeks was studied. In addition, the influence of the repair process of a stab wound on tumor induction by DMNU was examined. From seven weeks after the first injection of DMNU, a loss of subependymal cells appeared along with a decrease in labeling indices in flash labeling with 3H-thymidine. From twenty weeks, gliomas began to appear. Their distribution was much denser in the vicinity of the depleted subependymal layer than in the periphery of the cerebral hemisphere, and corresponded to the distribution of labeled cells in the normal adult brain. Microtumors composed of less mature glial cells grew to histologically mature gliomas with the lapse of time. No effect of the stab wound was observed on the incidence, distribution or latency period of glioma development. From these results, it was concluded that DMNU-induced gliomas develop in close relation to cellular differentiation of target cells. It was assumed that mature gliomas are derived from less mature glial cells in the glioblastic (spongioblastic) stage or migrating neuroglias remaining in adult rat brains.

Stimulation of DNA synthesis in [formula omitted] 3T3 cells by peripheral nerve degenerating in vitro

The release of mitogenic substances from degenerating peripheral nerves was detected and characterized in vitro. Cultures of serum-starved, subconfluent Balb c 3T3 cells were exposed 24 h to myelinated peripheral nerve fascicles, with [ 3H]thymidine added during the last 3 h. Cells exposed to peripheral nerves incorporated twice as much [ 3H]thymidine as control cultures without nerves ( P < 0.005). Autoradiography showed a graded decrease in labeling index with increasing distance from nerves. The mitogenic response varied in a dose-dependent manner with increasing nerve length. Also, the response varied according to the degree of myelination. Myelinated sciatic nerve fascicles caused greater incorporation of [ 3H]thymidine ( P < 0.005) than unmyelinated abdominal vagus nerves of similar size, suggesting myelin-derived growth factor activity. Evidence from other laboratories has led to the hypothesis that during peripheral nerve injury, myelin proteins are degraded by lysosome-derived acid proteinases yielding mitogenic polypeptide fragments. We report that the addition of the acid proteinase inhibitor, pepstatin, to the culture media caused a small but significant decrease ( P < 0.05) in the mitogenic effect of peripheral nerves. The work supports the concept that the cell proliferation accompanying Wallerian degeneration is stimulated by mitogens released by the injured nerve.

Tritiated thymidine autoradiographic study of the effects of inferior alveolar nerve resection on the proliferative compartments of the mouse incisor formative tissues

At 15, 30 and 60 days after denervation, 10 mice were injected (5 controls, 5 denervated) at each time period with 0.5 μCi/gm of [ 3H]-thymidine. Denervated teeth, after 15 and 30 days, were thinner, shorter and narrower with a chalky-white appearance; by 60 days, the teeth returned to their normal predenervated appearance. The responses of the proliferative compartments of the mouse incisor varied with time after denervation, and depended on whether comparison was made to sham controls or the control side of a denervated mouse. The pre-odontoblasts were most stable in terms of [ 3H]-thymidine labelling index with a significant decrease only at 60 days after surgery. This decrease may be due to an alteration of the inductive influence of the inner enamel epithelium and the nerve resection. The effects of nerve, resection on the inner enamel epithelium were a reduction in the labelling index on both control and denervated sides in comparisons to sham controls and a decrease in labelling index of the denervated side when compared to contralateral control. Significant differences between the control and denervated sides of experimental mice were evident 15 days after denervation in the outer enamel epithelium, inner enamel epithelium, and pulp fibroblasts. Indirect effects (i.e. chipping of the teeth) may mask neurotrophic influences. There also appears to be a homeostatic or compensatory mechanism for attenuation of the effects of denervation in continuously erupting teeth. The effects at the early time periods emphasize the need for a study of neurotrophic effects on the cellular compartments of the tooth in the period immediately following denervation.

Cell volume decrease during Friend leukemia cell differentiation.

Friend leukemia cells (GM86, clone 745) were induced to differentiate with dimethyl sulfoxide or butyric acid. The kinetics of induction were measured by cell growth, cell volume distributions, and [3H]thymidine incorporation. From the volume distributions, it was found that the rate of induction was both agent sensitive and concentration dependent. The changes in volume distributions occurred approximately 4 hr earlier with dimethyl sulfoxide induction relative to butyric acid induction. However, the changes with the butyric acid induction were more dependent on concentration. A decrease in labeling indices during the 12- to 20-hr time period was correlated to a decrease in mean cell volume and an increase in the proportion of G1 cells. After the 20-hr time period of induction, an increase in labeling indices and in the percentage of large cells was observed. The data suggest that a transient block of cells in G1 occurred between 12 to 20 hr, and that the early differentiation involved a volume decrease which was related to a redistribution of cell cycle stages. The study was also shown that the changes of cell volume are a rapid monitor to determine the early events of the differentiation process.

STUDIES ON THE DEPENDENCE OF MITOCHONDRIAL 11β- AND 18- HYDROXYLATION ON THE NUCLEAR AND MITOCHONDRIAL DNA SYNTHESIS DURING ACTH-INDUCED DIFFERENTIATION OF CORTICAL CELLS OF RAT ADRENALS IN TISSUE CULTURE

Abstract Replicative potential ([3H]TdR uptake as detected by autoradiography) of cultured fetal adrenocortical cells was correlated to their morphological and functional stage of differentiation. Simultaneously, replicative and differentiative phenomena within the mitochondrial compartment of the cells were evaluated. Addition of ACTH to the cultures resulted in a prompt cessation of growth (decrease in labelling indices). This occurred even before the differentiation of the cultures was evident at the level of cell ultrastructure and steroidogenesis. A burst of mitochondrial DNA synthesis (mitochondrial [3H]TdR accumulation) preceding mitochondrial proliferation was observed within the cells. Ethidium bromide (EB), an inhibitor of mitochondrial DNA and protein synthesis, inhibited the ACTH-induced mitochondrial numerical increase, and no differentiation of mitochondrial inner membranes took place. The steroid secretory pattern revealed an inhibition of the activation of steroid 11β- and 18-hydroxylases.

Studies on the dependence of mitochondrial 11β- and 18-hydroxylation on the nuclear and mitochondrial DNA synthesis during acth-induced differentiation of cortical cells of rat adrenals in tissue culture

Replicative potential ([ 3H]TdR uptake as detected by autoradiography) of cultured fetal adrenocortical cells was correlated to their morphological and functional stage of differentiation. Simultaneously, replicative and differentiative phenomena within the mitochondrial compartment of the cells were evaluated. Addition of ACTH to the cultures resulted in a prompt cessation of growth (decrease in labelling indices). This occurred even before the differentiation of the cultures was evident at the level of cell ultrastructure and steroidogenesis. A burst of mitochondrial DNA synthesis (mitochondrial [ 3H]TdR accumulation) preceding mitochondrial proliferation was observed within the cells. Ethidium bromide (EB), an inhibitor of mitochondrial DNA and protein synthesis, inhibited the ACTH-induced mitochondrial numerical increase, and no differentiation of mitochondrial inner membranes took place. The steroid secretory pattern revealed an inhibition of the activation of steroid 11β- and 18-hydroxylases.

Labeling index (tritiated thymidine) of the synovium of mice at different ages

The H 3-autoradiographic study of the cellular proliferative activity of the synovial tissue of the knee joints of aging mice reveals a significant decrease in labeling indices between 5 and 26 weeks of age. A low level persisted throughout the life span of the animal. These cellular proliferative changes exhibited by the synovium with increasing age are similar to those reported for other skeletal associated tissues. Labeling appeared to be restricted to the surface cells of the synovial lining.

Cytokinetics of neonatal cerebellar neurons in rats as studied by the "complete H3-thymidine labelling" method.

The development of rat cerebellum was studied by the complete 3H-thymidine labelling method which provides a 100% labelling of cell nuclei at the time of birth. The decrease in labelling index and intensity after birth was studied for a total of 42 days in order to compare proliferative activity of the different cell populations.

The cell proliferative activity of parodontal tissues in aging mice

One hundred and thirty-eight short-lived mice of the Brookhaven National Laboratory inbred strain of Swiss albino 5, 26, 52, 78 weeks of age were used to determine the normal proliferative activity of the parodontal tissues comprising the mesial aspects of the maxillary 1st molars and the associated changes concomitant with aging. Labelling indices were obtained following grain-counting of autoradiographs. Each animal received 1 μCi of tritiated thymidine per gram body weight 1 hr prior to death. Five micron sections were cut from paraffin blocks prepared routinely following EDTA decalcification. Results showed that the order of proliferative activity was similar at all ages. The proliferative activity was highest in gingival epithelium while all other tissue compartments were significantly lower. In lessening order, these were 4 connective tissue compartments, 2 osteogenic layers and lastly the cementoblastic layer. A progressive decrease in labelling indices to 52 weeks of age was generally observed with increasing age. This was subsequently followed by a rise in the number of labelled cells, especially in the gingival epithelium and connective tissue below the crevicular region. A survey of the tissues for inflammatory reactions revealed increased incidence with advancing age, especially at 78 weeks of age. The crevicular region of the epithelium, the connective tissue below and the periapical periodontium were the usual inflammatory sites. It was concluded that increased labelling observed in animals older than 52 weeks of age was associated with increased incidence of inflammation at susceptible sites coincidental with histological age changes in parodontal tissues.

The initiation of limb bud outgrowth in the embryonic chick

Changes in the rate of cell division that occur during the initiation of outgrowth of the embryonic chick wing have been investigated by determination of changes in the labeling index in the wing region and the flank region. It has been found that the labeling index changes very little in the wing region between stage 16 and stage 20, but decreases rapidly in the flank region during the same period. The decrease in labeling index in the flank region results from a change in the proliferative index in that region from 100% at stage 16 to 75% at stage 19. This change in the proliferative index in the flank cannot be easily correlated with differentiative changes in the flank, but correlates very well with morphological changes.

Preservation and long-term storage of Feulgen-stained mouse tissues for subsequent autoradiography.

Tissue of the jejunal crypts of mouse intestine was fixed for 24-48 hr in acetic-ethanol, 1:3, and stained en bloc by the Feulgen reaction. The stained preparations were then stored 4 mo at -25 C in either 45% acetic acid alone or in dimethyl sulfoxide (DMSO) or glycerol to which 45% acetic acid had been added to make 15% of the total volume. Such storage preserved not only the stain but allowed autoradiographs to be made. No loss of silver grains or a decrease of labeling index was observed. The procedures are equally successful when used with double-labeling experiments. Solid, transplantable, experimental carcinomas can be preserved in a manner identical to that suggested for the epithelial cells of the crypts.