Increase In Labeling Index Research Articles

PP - HISTOPATHOLOGICAL AND IMMUNOHISTOCHEMICAL ANALYSIS OF WNT/β-CATENIN SIGNALING PATHWAY PROTEINS IN ACTINIC CHEILITIS

Actinic cheilitis (AC) is a potentially malignant disorder of the lips that is caused by chronic exposure to ultraviolet radiation. The Wnt/β-catenin signaling pathway - also named the canonical signaling pathway - acts in cell proliferation and is involved with tumor development and progression. Some of these molecules have already been identified in AC. Thus, the aim of this study was to comparatively evaluate the histopathological features and the presence of Wnt/β-catenin signaling pathway proteins in AC. For this purpose, immunohistochemical reactions against Wnt1, Wnt5a, β-catenin, axin, APC and cyclin D1 were performed and evaluated in accordance with histopathological epithelial dysplasia grading, using the World Health Organization classification and the binary classification system. The results showed positivity for Wnt 1 (96.7%), cyclin D1 (75.4%), APC (96.7%), axin (95%) and cytoplasmic β-catenin staining (81.9%) and negativity for Wnt 5a (100%). Furthermore, Wnt1, cyclin D1, APC and axin showed an increased labeling index related with the degree of epithelial dysplasia using the binary system. In conclusion, it was possible to state that Wnt 1, cyclin D1 and β-catenin seems to act as tumor promoters by activating the canonical signaling pathway, whereas APC and axin seems to act as tumor promoters by activating the non-canonical signaling pathways.

Mantle cell lymphoma shows three morphological evolutions of classical, intermediate, and aggressive forms, which occur in parallel with increased labeling index of cyclin D1 and Ki‐67

Although the 2008 World Health Organization classification defines two subtypes of mantle cell lymphoma (MCL), classical and aggressive, we often encounter MCL with both features in the same site. We named this feature "MCL with focal aggressive form (intermediate MCL)". In the present study, we reclassified 237 patients with cyclin D1 (CCND1)-positive MCL on the basis of the concept of intermediate MCL, and analyzed the correlation of this reclassification with immunohistochemical detection of CCND1, Ki-67, p53, p27(Kip1), and p21(WAF/Cip1). The median overall survival was 77, 31, and 18 months for classical, intermediate, and aggressive MCL, respectively, showing a statistically significant difference (P < 0.0001). The expression levels of CCND1, Ki-67, p53, and p21(WAF/Cip1) in aggressive MCL (mean 80.1 +/- 27.8%, 73.7 +/- 28.9%, 31.0 +/- 69.0%, and 10.4 +/- 24.8%, respectively) were higher than those in classical MCL (mean 58.1 +/- 36.7%, 25.2 +/- 25.5%, 6.5 +/- 24.3%, and 2.5 +/- 13.0%, respectively) and intermediate MCL (mean 75.7 +/- 31.4%, 30.8 +/- 33.3%, 21.0 +/- 57.4%, and 4.8 +/- 16.5%, respectively). Significantly different levels of Ki-67 and p21(WAF/Cip1) were only recognized between intermediate and aggressive (P < 0.05 and P < 0.0001, respectively), whereas those of CCND1 and p53 were only between classical and intermediate (P < 0.0001 and P < 0.05, respectively). There were no significant differences in p27(Kip1) among the three groups. The subsequent discriminant analysis with independent prognostic factors clearly demonstrated that the morphological evolution of MCL occurs in parallel with increased labeling index of CCND1 and Ki-67. The diagnosis of intermediate MCL thus proved to be of major significance and should enable the design of more tailored therapies.

Melatonin and vitamin C administration ameliorate diazepam‐induced oxidative stress and cell proliferation in the liver of rats

Oxidative stress is a likely molecular mechanism in long-term diazepam administration. The benefits of antioxidants (melatonin and vitamin C) against diazepam-induced cell proliferation, DNA synthesis and oxidative damage were investigated in this study. Four equal-sized groups of male rats [control, diazepam (3 mg/kg), diazepam plus melatonin (5 mg/kg) and diazepam plus vitamin C (50 mg/kg)] were used. Levels of lipid peroxides (LPO), superoxide dismutase (SOD) activity and glutathione (GSH) concentration were measured in tissue homogenates. Cell proliferation and rate of DNA synthesis were detected by autoradiography. Results documented increased labelling index, (3)H-thymidine incorporation (DNA synthesis), LPO plus decrease in GSH levels and SOD activity in livers of diazepam-administered rats versus those of controls. When melatonin and vitamin C were given to diazepam-administered rats, they almost attenuated the increase of labelling index, DNA synthesis and LPO, and restored the levels of GSH and SOD activity. These results suggest long-term hazard in use of drugs such as diazepam; they may be toxic and damage terminates in complex liver damage. Furthermore, melatonin and vitamin C may be useful in combating free radical-induced liver injury resulting from hazard and/or repeated diazepam administration.

Effect of dentin depth on hybridization quality using different bonding tactics in vivo

Objectives Incomplete resin infiltration and polymerization of adhesive contributed to nanoleakage formation. This study tested the null hypothesis that adoption of different bonding tactics and dentine depth will not affect hybridization quality in vivo. Methods Class V cavities were prepared on the labial/buccal surface of monkey teeth. They were bonded by Single Bond (a two-step total-etch adhesive), Clearfil SE Bond (a two-step self-etch adhesive), or Clearfil S 3 Bond (an all-in-one self-etch adhesive). Combined nanoleakage analysis and quantitative immunolabeling evaluation were carried out in the hybrid layer formed in both cervical superficial and deep dentine. Results Single Bond showed reticular and spotted nanoleakage while Clearfil SE Bond and Clearfil S 3 Bond presented only a spotted one. While Single Bond showed increased concentration of labeling of type I collagen within the deep part of the hybrid layer, two self-etch adhesives—Clearfil SE Bond and Clearfil S 3 Bond revealed a homogeneous labeling pattern, even if the latter presented a significantly increased labeling index in deep dentine. Conclusions Different bonding tactics showed different nanoleakage patterns and immunolabeling index, and was influenced by dentine depth at different levels i n vivo.

Neonatal porcine endometrial development and epithelial proliferation affected by age and exposure to estrogen and relaxin

In the pig, temporospatially regulated proliferation of uterine luminal (LE) and glandular (GE) epithelium between birth (postnatal day = PND 0) and PND 15 is essential for success of endometrial development. Exposure of gilts to estrogen (E) or relaxin (RLX) during this period disrupts uterine development, and neonatal E exposure can compromise adult uterine function. Neonatal uterotrophic effects of E and RLX, administered for 2 days beginning on PND 12, can be inhibited with the antiestrogen ICI 182,780 (ICI) indicating crosstalk between RLX and E signaling systems. Here, objectives were to determine effects of: (study 1) neonatal age and (study 2) exposure to E, RLX, and ICI on porcine neonatal uterine histoarchitecture and patterns of epithelial cell proliferation as reflected by proliferating cell nuclear antigen labeling index. In study 1, uteri were obtained on PND 0, 3, 6, 9, 12 and 15. Glandular epithelium, absent at birth, was observed by PND 3. Overall, epithelial labeling index increased from birth to PND 3, declined from PND 6–9 in LE and GE, and increased to PND 15 in GE. In study 2, uteri were collected on PND 14 after administration of vehicle, E, or RLX for 2 days, or following pretreatment with ICI. Alone, E was uterotrophic and adenogenic and increased labeling index in both LE and GE. Both RLX and ICI increased proliferation in GE. Effects of E and RLX were attenuated by ICI, providing further support for crosstalk between these signaling systems in the developing neonatal porcine endometrium.

Enhanced Expression of p53 Protein in Pityriasis Rubra Pilaris

p53 is a phosphoprotein, known since 1992 as a “guardian” of the genome (1, 2). A mutation in the p53 gene coding this protein, which activation protects the organism from tumourigenesis, has been found in more than half of all human tumour types (3). DNA damaging agents induce p53 protein activity, which leads to cell cycle arrest in the G1 or G2 phase and, in the case of ineffective DNA repair, apoptosis is initiated (4). p53 immunoreactivity has also been found in non-tumourigenic inflammatory skin diseases, e.g. psoriasis, chronic dermatitis, lupus erythematosus and lichen planus (5), often with contro-versial results. p53 is also detectable in normal skin, using various methods of antigen retrievals, especially after ultraviolet (UV) exposure, but at very low levels (6).Pityriasis rubra pilaris (PRP) is a rare hyperkeratotic, papulo-squamous skin disorder with unknown aetio-logy. Although the first case was described in 1828, until now the pathogenesis of this rare disorder is still a matter for debate. A few possible mechanisms are considered: abnormal vitamin A metabolism in the skin, association with internal malignancies, immune system dysregulation (PRP often appears simultaneously with autoimmune diseases) and associations with infections, particularly human immunodeficiency virus. Hereditary forms of PRP are very rare (7, 8). Experimental stu-dies showed that epidermal cells in PRP lesions have increased labelling index compared with normal skin keratinocytes, similarly to findings in psoriatic skin (9, 10). PRP presents with some histological features simi-lar to psoriasis, especially hyperkeratosis, parakeratosis and acanthosis (7, 8, 11), which could result from cell cycle disturbances in the keratinocytes.The present study was undertaken to evaluate ex-pression of p53 protein in the lesional skin of patients with type I classic PRP (confirmed by histological examination).PATIENTS AND METHODS

Estradiol receptor binding to the epithelium of uterine lumen and glands: region- and time-related changes during preimplantation and periimplantation periods studied by autoradiography.

The presence and changes of estradiol nuclear binding and related functions in uterine luminal and glandular epithelium were studied before and after blastocyst implantation using receptor autoradiography with (3)H-estradiol-17beta in association with (3)H-thymidine incorporation and immunocytochemical binding of antibody to estrogen receptor ER-alpha. (3)H-estradiol nuclear binding is present but variable during days 1.5-7.5 of pregnancy. Sites of strong nuclear binding of (3)H-estradiol exhibit strong immunocytochemical staining with ER-alpha antibody. Qualitative and quantitative evaluation of autoradiograms reveal that there is a general increase of nuclear (3)H-estradiol binding during the first 3 days after fertilization in both luminal and glandular epithelium. The binding of estradiol is stronger in glandular epithelium from day 2.5 to day 7.5, paralleled by a rise in (3)H-thymidine incorporation on day 2.5. By comparison, in the epithelium of the uterine lumen (3)H-estradiol nuclear binding is low, but relatively high in epithelial cells at lateral branching of the lumen where the increase in (3)H-estradiol binding corresponds to an increased labeling index with (3)H-thymidine. A highly differentiated binding of (3)H-estradiol to luminal and glandular epithelium was demonstrated with region- and time-specific changes of related effects on cell proliferation, differentiation, and secretion, probably involving involution and remodeling. The strong (3)H-estradiol binding to glandular epithelium suggests that estradiol exerts pronounced effects on glandular activities in the periimplantation period.

Chemoprevention of tea on colorectal cancer induced by dimethylhydrazine in Wistar rats.

AIM:To investigate the chemopreventive effects of green tea and tea pigment on 1,2-dimethylhydrazine (DMH)-induced rat colorectal carcinogenesis.METHODS:Male weaning Wistar rats were randomly allocated into four groups.Rats in the positive control group were given a(c)s.c. injection of DMH, once a week for ten weeks;rats in tea treated groups, with the same DMH treatment as in the positive group, received 2% green tea and 0.1% tea pigments; rats in the negative control group were given s.c.injection of the same volume of saline as well as DMH in the positive group. Animals were sacrified and necropsied at the end of week 16 and week 32.RESULTS:Aberrant cryptic foci (ACF) were formed in animals in DMH-treated groups at the end of week 16. Compared to the DMH group, green tea and tea pigments groups had less ACF (148.25 and 204.25, respectively, P<0.01). At the end of week 32, all rats in DMH group developed large intestinal tumors. The results also showed that DMH increased labeling index (LI) of proliferating cell nuclear antigen (PCNA) of intestinal mucosa and the expression of ras-p21. However, in the tea-treated groups, PCNA-LI was significantly reduced as compared with the positive control group (36.63 and 40.36 in the green tea group and tea pigment group, respectively, at the end of the experiment,P<0.01).ras-p21 expression was also significantly reduced (2.07 and 2.36 in the colon tumors of rats in the green tea group and tea pigments group, respectively at the end of the experiment, P<0.01). Furthermore, green tea and tea pigment inhibited the expression of Bcl-2 protein (2, 5, 1, 0 and 2, 4, 1, 0,respectively, at the end of the experiment P<0.01), and induced expression of Bax protein (0,1,3,4 and 0,1,4,3, respectively, P <0.01).CONCLUSION:Chinese green tea drinking inhibited ACF and colonic tumors formation in rats, which showed that tea had a significant chemopreventive effect on DMH-induced colorectal carcinogenesis. Such effects may be due to suppression of cell proliferation and induction of apoptosis in the intestinal crypts.

The bone marrow plasma cell labeling index by flow cytometry.

The bone marrow plasma cell labeling index is the most important prognostic indicator for patients with multiple myeloma. Traditionally, this test has been performed as a two color immunofluorescent microscope technique which is time consuming and requires a degree of subjectivity in its interpretation. We have assessed various adaptations of this method to flow cytometry. A bromodeoxyuridine method has been compared with a propidium iodide DNA method to detect cells in S phase and CD38-FITC has been compared with CD38-FITC + CD138-FITC and CD38-biotin + streptavidin FITC to identify plasma cells. The mean channel fluorescent intensity of the plasma cell peaks for each of these markers was 12. 7, 17.4 and 35.3 respectively demonstrating the superiority of CD38-biotin + streptavidin FITC. Analysis after propidium iodide staining provided a good correlation with the slide technique (r = 0. 71; P < 0.0001) but the bromodeoxyuridine method did not correlate with the slide method (r = 0.09; P = NS). The labeling index values obtained from either of the flow methods were greater than the microscopic method. Thus a labeling index of >4% will replace the traditional >1% threshold for identifying patients with a significantly increased labeling index. The advantages of the new method are that it takes less time to perform, is more objective and provides additional data on ploidy and cell cycle status.

Cell proliferation induced by 3,3',5-triiodo-L-thyronine is associated with a reduction in the number of preneoplastic hepatic lesions.

Previous studies have suggested that liver cell proliferation is fundamental for the growth of carcinogen-initiated cells. To gain further information on the association between cell proliferation and hepatocarcinogenesis, we have examined the effect of the hormone 3,3',5-triiodo-L-thyronine (T3), a strong liver mitogen, on the growth of diethylnitrosamine (DENA)-induced hepatic lesions positive for the placental form of glutathione S-transferase (GSTP). Two weeks after a single initiating dose of DENA (150 mg/kg), cycles of liver cell proliferation were induced in male Fischer rats by feeding a T3-supplemented diet (4 mg/kg) 1 week/month for 7 months. Rats were killed at the end of the seventh cycle or 1 month later. Results indicate that, in spite of an increased labelling index, a 70% reduction in the number/cm(2) of GSTP-positive minifoci occurred in T3-treated rats. A decrease in the number of GSTP-positive foci was also observed in T3-treated rats killed 1 month after the last exposure to the hormone (40, versus 67 foci/cm(2) in controls), indicating that the reduction was not due to an inhibitory effect on GSTP exerted by the concomitant presence of T3. In a second series of experiments where DENA-treated rats were fed T3 for 1 week and then subjected to the resistant hepatocyte (RH) model, it was found that T3 treatment prior to promotion resulted in a decrease in the number of GSTP-positive foci (16 GSTP(+) foci/cm(2) in T3-fed animals versus 45 in the control group). The results indicate that cell proliferation associated with T3 treatment: (i) reduces the number of carcinogen-induced GSTP-positive lesions; (ii) does not exert any differential effect on the growth of the remaining foci; (iii) inhibits the capacity of putative DENA-initiated cells to be promoted by the RH model. Data suggest that cell proliferation may not necessarily represent a stimulus for the growth of putative preneoplastic lesions.

Effect of vascular endothelial growth factor on hepatic regenerative activity following partial hepatectomy in rats

Background/Aims: Vascular endothelial growth factor (VEGF) is an angiogenic factor with a growth-promoting effect that is thought to be restricted to vascular endothelial cells. Its essential role during liver regeneration has yet to be determined. The aim of this study was to document the effect of exogenous VEGF administration on liver regeneration in rats undergoing submaximal hepatic resections. Methods: Adult male Sprague-Dawley rats ( n=4/group) undergoing 30% partial hepatectomy were administered 200 ng VEGF 165 intravenously and were sacrificed at 24, 36, and 48 h postoperatively. Liver regeneration was monitored by measuring the restituted liver mass, proliferating cell nuclear antigen (PCNA) immunostaining, and hepatic PCNA protein by Western blot. Results: Changes in restituted liver mass 48 h postsurgery were more prominent, but did not differ statistically between VEGF-treated and control rats (47% vs 29%; p<0.06). Nevertheless, PCNA immunostaining showed increased labeling index of hepatocytes, apparent at 36 and 48 h after partial hepatectomy (38% vs 18% [ p<0.04] and 42% vs 11% [ p<0.02], respectively). Hepatic PCNA proteins measured by Western blot showed a 3-fold increase in VEGF-treated rats 48 h postsurgery compared with controls ( p<0.01). Conclusion: Exogenous VEGF administration early after partial hepatectomy stimulates liver regeneration in rats. Whether or not VEGF 165 is a direct mitogen for hepatocytes remains to be determined.

Increased Thyroid Epithelial Cell Proliferation in Toxic Thyroid Nodules

Most toxic thyroid nodules (TTN) result from clonal expansion of a single cell caused by a somatic mutation in the thyrotropin (TSH) receptor, the Gsalpha protein, or yet unknown proteins. Expanding a single cell into a TTN with thousands of cells suggests a prolonged increase in proliferation compared to nonaffected surrounding cells. To test this hypothesis, we evaluated cell proliferation in TTN. Tissue from 20 TTN and their surrounding normal thyroid tissue was studied for the occurrence of the proliferating cell nuclear antigen (PCNA) and Ki-67 epitope as markers for cell proliferation. The labeling index (number of labeled cells versus total cell number) for nodular and surrounding tissue was calculated. Nineteen samples were evaluated for PCNA immunohistochemistry. In 16 TTN, a significant (p< or =0.05%) up to 3-fold increase in the labeling index for PCNA was detectable. In only 3 toxic nodules (2 without a detectable TSH receptor or Gsalpha protein mutation), we found no significant difference in the labeling index compared to the surrounding tissue. Because labeling for KI-67 was much lower, only 16 toxic thyroid nodules were quantified. Twelve of these showed significantly (p< or =0.05%) increased labeling indices. The increase of the labeling index for both markers was similar for histologically defined adenoma versus adenomatous nodule or nodules with or without TSH receptor mutation or clonal versus polyclonal origin of toxic nodules studied. These findings are evidence that an increased thyroid epithelial cell proliferation is a uniform feature common to most TTNs, independent of their histopathological or molecular characteristics. Although increased proliferation in many TTNs is very likely the result of TSH receptor mutations, the cause of increased proliferation in TTN without a mutation is unknown.

Prognostic significance of telomerase in brain tumors

Telomeres help to maintain chromosomal stability and integrity. They are progressively lost, conditioning aging and mortality. Telomerase enzyme activation stabilizes the telomere length, permitting the proliferation of cancer cells. Four papers are reviewed dealing with increased expression of telomerase associated with increased labelling index of MIB-1 (Ki-67) and malignancy in astrocytomas, ependymomas, and oligodendrogliomas. Telomerase expression can be utilized for differentiating malignant from benign tumors with histological resemblance. The significance of telomerase in tumor progression is discussed within the field of brain neoplasias.

Subchronic Inhalation Studies of Styrene in CD Rats and CD-1 Mice

Groups of 10 male and 10 female Charles River (CRL) CD (Sprague-Dawley-derived) rats were exposed to styrene vapor at 0, 200, 500, 1000, or 1500 ppm 6 hr per day 5 days per week for 13 weeks. Styrene had no effect on survival, hematology, or clinical chemistry. Males at 1500 ppm weighed 10% less after 13 weeks and males and females at 1000 and 1500 ppm consumed more water than controls. Histopathologic changes were confined to the olfactory epithelium of the nasal mucosa. Groups of 20 male and 20 female CRL CD-1 and B6C3F1 mice were exposed to styrene vapor at 0, 15, 60, 250, or 500 ppm 6 hr per day 5 days per week for 2 weeks. Mortality was observed in both CD-1 and B6C3F1 mice exposed to 250 or 500 ppm; more female mice, but not males, died from exposure to 250 ppm than from 500 ppm. Groups of 10 male and 10 female CRL CD-1 mice were exposed to styrene vapors at 0, 50, 100, 150, or 200 ppm 6 hr per day 5 days per week for 13 weeks. Two females exposed to 200 ppm died during the first week. Liver toxicity was evident in the decedents and in some female survivors at 200 ppm. Changes were observed in the lungs of mice exposed to 100, 150, or 200 ppm and in the nasal passages of all treatment groups, those exposed to 50 ppm being less affected. Satellite groups of 15 male rats and 30 male mice were exposed as described above for 2, 5, or 13 weeks for measurement of cell proliferation (BrdU labeling). No increase in cell proliferation was found in liver of rats or mice or in cells of the bronchiolar or alveolar region of the lung of rats. No increase in labeling index of type II pneumocytes was seen in mouse lungs, while at 150 and 200 ppm, an increased labeling index of Clara cells was seen after 2 weeks and in occasional mice after 5 weeks. Large variations in the labeling index among animals emphasize the need for large group sizes. For nasal tract effects, a NOAEL was not found in CD-1 mice, but in CD rats, the NOAEL was 200 ppm. For other effects, the NOAEL was 500 ppm in rats and 50 ppm in mice.

A transgenic mouse model that recapitulates the clinical features of both neonatal and adult forms of the skin disease epidermolytic hyperkeratosis

Keratins are the major structural proteins of keratinocytes, which are the most abundant cell type in the mammalian epidermis. Mutations in epidermal keratin genes have been shown to cause severe blistering skin abnormalities. One such disease, epidermolytic hyperkeratosis (EHK), also known as bullous congenital ichthyosiform erythroderma, occurs as a result of mutations in highly conserved regions of keratins K1 and K10. Patients with EHK first exhibit erythroderma with severe blistering, which later is replaced by thick patches of scaly skin. To assess the effect of a mutated K1 gene on skin biology and to produce an animal model for EHK, we removed 60 residues from the 2B segment of HK1 and observed the effects of its expression in the epidermis of transgenic mice. Phenotypes of the resultant mice closely resembled those observed in the human disease, first with epidermal blisters, then later with hyperkeratotic lesions. In neonatal mice homozygous for the transgene, the skin was thicker, with an increased labeling index, and the spinous cells showed a collapse of the keratin filament network around the nuclei, suggesting that a critical concentration of the mutant HK1, over the endogenous MK1, was required to disrupt the structural integrity of the spinous cells. Additionally, footpad epithelium, which is devoid of hair follicles, showed blistering in the spinous layer, suggesting that hair follicles can stabilize or protect the epidermis from trauma. Blisters were not evident in adult mice, but instead they showed a thick, scaly hyperkeratotic skin with increased mitosis, resulting in an increased number of corneocytes and granular cells. Irregularly shaped keratohyalin granules were also observed. To date, this is the only transgenic model to show the typical morphology found in the adult form of EHK.

The effect of a nucleotide-nucleoside solution on hepatic regeneration after partial hepatectomy in rats

After hepatectomy, purine and pyrimidine metabolism is a key process in the synthesis of DNA and RNA and maintaining cellular energy metabolism. The purpose of this study is to evaluate changes in blood purine and pyrimidine levels after partial hepatectomy and the effect of purine and pyrimidine nucleoside solution injection on hepatic regeneration under the hypothesis that the rat after partial hepatectomy requires substrates for salvage nucleotide synthesis and changes blood nucleoside and nucleobase levels. Blood levels of nucleotides, nucleosides, and nucleobase by high-performance liquid chromatography method and liver ATP level by enzymatic analysis, and the effect of preoperative injection of nucleoside solution (OG-VI) on hepatic regeneration ratio and hepatocytes DNA synthesis, were assessed in rats after 70% partial hepatectomy. Decreased liver adenosine triphosphate and increased plasma xanthine and hypoxanthine after partial hepatectomy indicated an increase in catabolism of purine nucleotides in regenerating liver. Plasma thymidine and cytidine levels increased, then returned to the prevalue, suggesting that the thymidine and cytidine pool was enlarged. OG-VI increased labeling indices of hepatocytes at postoperative d 1 (POD) and hepatic regeneration ratio at POD 14. Blood purine nucleobase and pyrimidine nucleoside levels change after partial hepatectomy and preoperative supply of nucleoside solution is effective for increasing hepatocytes DNA synthesis and hepatic regeneration after partial hepatectomy.

Epithelial cell proliferation during colonic chemical carcinogenesis in the rat.

To define the significance of alterations in epithelial cell proliferation as a marker of high risk mucosa for colorectal cancer, we examined cell proliferative events in the colonic mucosa during chemical carcinogenesis using in vitro bromodeoxyuridine labelling and by analysing serial colonoscopic biopsy specimens from dimethylhydrazine (DMH)-treated rats. In both the rectum and flexure of the colon, an increased labelling index of colonic epithelial cells, an upward extension of the proliferating zone and an upward shift of the major area of DNA synthesis of epithelial cells were observed during DMH-induced colonic carcinogenesis in rats. These changes preceded the development of the colonic tumour and were observed in endoscopically normal rectal mucosa where the tumour was absent. We confirmed the altered cell proliferative events preceding the development of the tumour by examining serial colonoscopic biopsies. The results suggest that these alterations are features that identify premalignant colonic mucosa in DMH-treated rats.

Caecal and colonic tissue structure and proliferation as influenced by adaptation period and indigestible polysaccharides

Growing rats were fed experimental diets differing in the type of polysaccharide, for adaptation periods of either 9, 19, 33, 47, or 61 days. The experimental diets contained 8% of either cellulose (CEL), guar gum (GG), pectin (PEC), or resistant starch (RS). A fibre-free diet (FF), containing normal corn starch only, acted as a control diet. Caecal and colonic proliferation activities, in terms of labelling indexes, were significantly higher at the initiation of the experiment (day 0) than observed after the respective adaptation periods. Differences between the adaptation periods were small and inconsistent. Epithelial structure and cellular proliferation depended on the diet and diet effects appeared to be most pronounced in the caecum and least in the distal colon. Epithelial cell migration rates were increased by diet RS and to some extent by diet GG as compared to diet CEL. The more fermentable diets (diets GG and PEC) increased the labelling indexes in the caecum, proximal colon and, to some extent, the distal colon compared to the diet with microbially inert fibre (diet CEL). In the caecum, the increased labelling index was associated with an expansion of the proliferation zones. The expansion of the proliferation zone appeared to take place towards the crypt base rather than towards the crypt mouth. Only small changes in epithelial structure and proliferation were observed after day 9, indicating that adaptation to the experimental diets persisted for as long as the rats received the diets.

Different features of Ca 2+ oscillations in differentiated and undifferentiated hepatocyte doublets

Cytosolic free Ca 2+ ([Ca 2+] i) oscillations are postulated to play a critical role in cellular proliferation. By using doublets of normal rats (NR) and those 18 hours after two-thirds hepatectomy (PHR), we investigated cytosolic free Ca 2+ ([Ca 2+] i) responses in liver regeneration. Normal rat hepatocyte doublets that retain their bile canaliculi are polarized and well differentiated. PHR doublets, which also retain their bile canaliculi, were characterized as undifferentiated by (1) decreased canalicular secretion of fluorescein-isothiocyanate-labeled glycocholate; (2) increased labeling index of hepatocytes in BrdU staining (∼30%); and (3) impaired transfer of fluorescent dye injected into one cell of the pair to the other. Addition of phenylephrine to NR and PHR doublets in the presence of extracellular Ca 2+ resulted in [Ca 2+] i oscillations or a nonoscillatory-sustained increase in [Ca 2+] i, followed by a gradual return to the baseline. Extracellular Ca 2+ was not required for [Ca 2+] i, oscillations but was necessary for a sustained increase in [Ca 2+] i. Simultaneous addition of prazosin, al-receptor blocker, to doublets immediately abolished these [Ca 2+] i, responses. The [Ca 2+] i level in each of the adjacent cells was synchronous in sustained increase in [Ca 2+] i, but asynchronous in [Ca 2+] i oscillations. As the phenylephrine concentration was increased (1 to 100 μmol/L), oscillations were replaced by a sustained increase in [Ca 2+] i in NR doublets. In contrast, in PHR doublets, oscillations remained, whereas the frequency of oscillations increased in a dose-dependent manner. These results indicate that the mechanisms of phenylephrineevoked [Ca 2+] i responses are different in differentiated and undifferentiated doublets and that the frequency modulation of [Ca 2+] i oscillations may be involved in the intracellular signal transduction in the cellular proliferation process during liver regeneration.

Different features of ca2+ oscillations in differentiated and undifferentiated hepatocyte doublets

Cytosolic free Ca2+ ([Ca2+]i) oscillations are postulated to play a critical role in cellular proliferation. By using doublets of normal rats (NR) and those 18 hours after two-thirds hepatectomy (PHR), we investigated cytosolic free Ca2+ ([Ca2+]i) responses in liver regeneration. Normal rat hepatocyte doublets that retain their bile canaliculi are polarized and well differentiated. PHR doublets, which also retain their bile canaliculi, were characterized as undifferentiated by (1) decreased canalicular secretion of fluorescein-isothiocyanate-labeled glycocholate; (2) increased labeling index of hepatocytes in BrdU staining (approximately 30%); and (3) impaired transfer of fluorescent dye injected into one cell of the pair to the other. Addition of phenylephrine to NR and PHR doublets in the presence of extracellular Ca2+ resulted in [Ca2+]i oscillations or a nonoscillatory-sustained increase in [Ca2+]i followed by a gradual return to the baseline. Extracellular Ca2+ was not required for [Ca2+]i oscillations but was necessary for a sustained increase in [Ca2+]i. Simultaneous addition of prazosin, alpha 1-receptor blocker, to doublets immediately abolished these [Ca2+]i responses. The [Ca2+]i level in each of the adjacent cells was synchronous in sustained increase in [Ca2+]i but asynchronous in [Ca2+]i oscillations. As the phenylephrine concentration was increased (1 to 100 mumol/L), oscillations were replaced by a sustained increase in [Ca2+]i in NR doublets. In contrast, in PHR doublets, oscillations remained, whereas the frequency of oscillations increased in a dose-dependent manner. These results indicate that the mechanisms of phenylephrine-evoked [Ca2+]i responses are different in differentiated and undifferentiated doublets and that the frequency modulation of [Ca2+]i oscillations may be involved in the intracellular signal transduction in the cellular proliferation process during liver regeneration.