DNA Labeling Index Research Articles

Single-stranded DNA (ssDNA) Labeling Index Is Related to Risk Grade in Patients with Gastrointestinal Stromal Tumors.

The main objective of the present study was to evaluate the significance of apoptosis in the Fletcher's risk classification for gastrointestinal stromal tumors (GISTs). Apoptotic cells were identified by immunostaining for single-stranded DNA (ssDNA). We assigned each GIST to one of four risk groups: very low risk, n=32; low risk, n=53; intermediate risk, n=15; high risk, n=6). The mean ssDNA labeling index for each group was 8.0±44.2, 20.1±86.5, 18.7±38.6 and 5.7±5.7, respectively. Fletcher's risk classification for GISTs correlated significantly with the ssDNA labeling index (p=0.002). The ssDNA labeling index and Ki-67 labeling index were the most significant factors corresponding to the risk grade of GISTs. These findings suggest that the ssDNA labeling index might be useful for predicting aggressive biological behavior of GISTs.

Thyroid hormone induces cyclin D1 nuclear translocation and DNA synthesis in adult rat cardiomyocytes

Although mammalian cardiomyocytes lose their proliferative capacity after birth, there is evidence that postmitotic cardiomyocytes can proliferate provided that cyclin D1 accumulates in the nucleus. Here we show by Northern blot, Western analysis, and immunohistochemistry that 3,5,3'-triiodothyronine (T3) treatment of adult rats caused an increase of cyclin D1 mRNA and protein levels. The increased cyclin D1 protein content was associated with its translocation into the nucleus of cardiomyocytes. These changes were accompanied by the re-entry of cardiomyocytes into the cell cycle, as demonstrated by increased levels of cyclin A, PCNA, and incorporation of bromodeoxyuridine into DNA (labeling index was 30.2% in T3-treated rats vs. 2.2% in controls). Entry into the S phase was associated with an increased mitotic activity as demonstrated by positivity of cardiomyocyte nuclei to antibodies anti-phosphohistone-3, a specific marker of the mitotic phase (mitotic index was 3.01/1000 cardiomyocte nuclei in hyperthyroid rats vs. 0.04 in controls). No biochemical or histological signs of tissue damage were observed in the heart of T3-treated rats. These results demonstrated that T3 treatment is associated with a re-entry of cardiomyocytes into the cell cycle and so may be important for the development of future therapeutic strategies aimed at inducing proliferation of cardiomyocytes.

Role of transforming growth factor-β1 (TGF-β1) in endotoxin-induced hepatic failure after extensive hepatectomy in rats

Postoperative infections after hepatectomy sometimes lead to fatal hepatic failure, but the mechanism of the hepatic failure is unclear. Wistar rats underwent 90% hepatectomy, and were then divided into three groups: (i) the SAL group, injected with normal saline; (ii) the LPS group, injected with lipopolysaccharide (LPS) every day for 1 week; and (iii) the LPS plus TGF-Ab (LPS+TGF-Ab) group, injected with LPS with anti-transforming growth factor-β1 (TGF-β1) antibody. We investigated survival rates, TGF-β1 expression in the liver, liver regeneration by proliferating cell nuclear antigen labeling index, hepatocyte apoptosis by single stranded DNA labeling index, and perisinusoidal fibrosis using Masson's trichrome staining. The LPS group (30.4%) had a significantly lower survival rate than the SAL group (84%) and tended to be lower than the LPS+TGF-Ab group (49.4%). Liver regeneration in the LPS group was significantly lower than in the other groups. In the LPS group, hepatocyte apoptosis and perisinusoidal fibrosis was significantly more remarkable, and TGF-β1 expression was significantly higher than in the SAL group. TGF-β1 enhanced by LPS plays an important role in the mechanism of hepatic failure by infections after hepatectomy, especially in inhibition of liver regeneration, and induction of hepatocyte apoptosis and perisinusoidal fibrosis.

Role of transforming growth factor-β1 (TGF-β1) in endotoxin-induced hepatic failure after extensive hepatectomy in rats

Postoperative infections after hepatectomy sometimes lead to fatal hepatic failure, but the mechanism of the hepatic failure is unclear. Wistar rats underwent 90% hepatectomy, and were then divided into three groups: (i) the SAL group, injected with normal saline; (ii) the LPS group, injected with lipopolysaccharide (LPS) every day for 1 week; and (iii) the LPS plus TGF-Ab (LPS+TGF-Ab) group, injected with LPS with anti-transforming growth factor-beta1 (TGF-beta1) antibody. We investigated survival rates, TGF-beta1 expression in the liver, liver regeneration by proliferating cell nuclear antigen labeling index, hepatocyte apoptosis by single stranded DNA labeling index, and perisinusoidal fibrosis using Masson's trichrome staining. The LPS group (30.4%) had a significantly lower survival rate than the SAL group (84%) and tended to be lower than the LPS+TGF-Ab group (49.4%). Liver regeneration in the LPS group was significantly lower than in the other groups. In the LPS group, hepatocyte apoptosis and perisinusoidal fibrosis was significantly more remarkable, and TGF-beta1 expression was significantly higher than in the SAL group. TGF-beta1 enhanced by LPS plays an important role in the mechanism of hepatic failure by infections after hepatectomy, especially in inhibition of liver regeneration, and induction of hepatocyte apoptosis and perisinusoidal fibrosis.

Growth index, assessed with Ki-67 and ssDNA labeling, is a significant prognosticator for patients who underwent curative resection for hepatocellular carcinoma

4084 Backgroud: The growth of tumor depends on the degree of imbalance between cell production and cell loss. Knowledge of such biological difference is of importance to understand aggressiveness of the cancer and to the management of cancer patients. This study was conducted to clarify a possible role of the modified prognosticator, Growth Index (GI), in patients who underwent curative resection for hepatocellular carcinoma (HCC). GI (%) was defined as a difference between a Ki-67 labeling index (%) and a single-stranded DNA (ssDNA) labeling index (%). Methods: HCC tissue specimens were obtained from 40 HCC patients (27 men and 13 women, range 45–80 years old) who underwent curative resection between April 1998 and March 2001. Immunohistochemical staining was perfomed using the avidin-biotin-peroxidase-complex method on formalin-fixed paraffin-embedded-tissues. Results: Ki-67 labeling index ranged from 0.20% to 29.10%, and ssDNA labeling index ranged from 0.22% to 4.20%. The GI ranged from -1.90% to 28.65% with a median of 3.73. GI was related with histologic grade (P = 0.014 by Kruskal-Wallis), pathologic T stage (P = 0.026 by Kruskal-Wallis), and intrahepatic metastasis (P = 0.048 by Mann-Whitney). The cumulative survival was poorer in patients with higher GI (≥ 3.73%, a median value) than those with lower GI (P = 0.014 by log-rank test). The univariate analysis demonstrated that GI, histologic grade, pathologic T stage, vascular invasion, intrahepatic metastasis, and age were significant factors affecting survival. The Cox multivariate analysis indicated that GI (P = 0.003) is an independent prognosticator along with intrahepatic metastasis (P = 0.003) and age (P = 0.018). Conclusions: Higher values of GI were significantly associated with histologically aggressive features of HCC, and GI was a significant independent prognosticator in HCC patients after curative resection. Assessing GI is feasible on routine paraffin sections and may provide additional prognostic information in those post-operative patients. No significant financial relationships to disclose.

Growth index, assessed with Ki-67 and ssDNA labeling, is a significant prognosticator for patients who underwent curative resection for hepatocellular carcinoma

4084 Backgroud: The growth of tumor depends on the degree of imbalance between cell production and cell loss. Knowledge of such biological difference is of importance to understand aggressiveness of the cancer and to the management of cancer patients. This study was conducted to clarify a possible role of the modified prognosticator, Growth Index (GI), in patients who underwent curative resection for hepatocellular carcinoma (HCC). GI (%) was defined as a difference between a Ki-67 labeling index (%) and a single-stranded DNA (ssDNA) labeling index (%). Methods: HCC tissue specimens were obtained from 40 HCC patients (27 men and 13 women, range 45–80 years old) who underwent curative resection between April 1998 and March 2001. Immunohistochemical staining was perfomed using the avidin-biotin-peroxidase-complex method on formalin-fixed paraffin-embedded-tissues. Results: Ki-67 labeling index ranged from 0.20% to 29.10%, and ssDNA labeling index ranged from 0.22% to 4.20%. The GI ranged from -1.90% to 28.65% with a median of 3.73. GI was related with histologic grade (P = 0.014 by Kruskal-Wallis), pathologic T stage (P = 0.026 by Kruskal-Wallis), and intrahepatic metastasis (P = 0.048 by Mann-Whitney). The cumulative survival was poorer in patients with higher GI (≥ 3.73%, a median value) than those with lower GI (P = 0.014 by log-rank test). The univariate analysis demonstrated that GI, histologic grade, pathologic T stage, vascular invasion, intrahepatic metastasis, and age were significant factors affecting survival. The Cox multivariate analysis indicated that GI (P = 0.003) is an independent prognosticator along with intrahepatic metastasis (P = 0.003) and age (P = 0.018). Conclusions: Higher values of GI were significantly associated with histologically aggressive features of HCC, and GI was a significant independent prognosticator in HCC patients after curative resection. Assessing GI is feasible on routine paraffin sections and may provide additional prognostic information in those post-operative patients. No significant financial relationships to disclose.

Chemoprevention of Colorectal Cancer by Grape Seed Proanthocyanidin Is Accompanied by a Decrease in Proliferation and Increase in Apoptosis

: Effects of proanthocyanidin (PA), procyanidin B-2 (B-2), and epigallocatechin gallate (EGCG) on azoxymethane (AOM)-induced colonic preneoplastic aberrant crypt foci (ACF) formation were investigated using F344 rats. The numbers of total ACF in rats treated with 0.002% PA and 0.05% B-2 were significantly decreased compared with the AOM alone group (control). Cell proliferation in the colon, as shown by proliferating cells nuclear antigen (PCNA), was also reduced in those treatments. The single-stranded DNA (ssDNA) labeling index, a marker for apoptosis, was significantly increased in 0.002% PA and 0.05% B-2 groups compared with control. Moreover, the numbers of CD11b/c+ cells (macrophages) and NKR-P1A+ cells (NK cells) in the all groups were significantly increased compared with control. In an in vitro study using rat colon cancer cell line RCN-9, PA, especially 5-10mer of PA (PA5/10), strong growth inhibition was shown. PA5/10 caused the most remarkable apoptosis as cleared by FACS analysis. These cells showed significantly increased caspase-3 activity. The results would suggest that the PA, especially PA5/10, might strongly enhance caspase-3 activity and cause apoptosis in cancer cells. PA at fairly low doses in the long term might serve as an effective means for preventing colon carcinogenesis.

Effects of Dexamethasone or Celecoxib on Biliary Toxicity after Hepatic Arterial Infusion of 5-Fluorodeoxyuridine in a Canine Model

Previous work has shown that in humans the dose-limiting toxicity for fluorodeoxyuridine [2-fluoro-5'-deoxyuridine (FdUrd)] when administered by hepatic arterial infusion is biliary sclerosis. The current study was undertaken to attempt to modify this toxicity in a canine model that has been demonstrated to closely mimic the clinical situation. Unlike previous studies using this model, in which animals were sacrificed after extensive fibrosis had already occurred, the current experiments were designed so that observations of pathology were made at an earlier time, when the initial inflammatory injury underlying the fibrotic process was still taking place. Implantable pumps were used to deliver FdUrd into the hepatic artery of animals at a rate of 0.3 mg/kg/day in the presence or absence of 10 mg/week dexamethasone or 100 mg/day of celecoxib for 35 days, at which time the animals were beginning to show signs of toxicity. After evaluation for radiological evidence of biliary obstruction, the animals were sacrificed and portions of their livers were processed for examination of microscopic pathology and 2-bromo-5'deoxyuridine labeling index. Dexamethasone treatment protected the animals from biliary sclerosis determined radiologically, further validating this model as being representative of the response in humans. Similarly the Cox-2 inhibitor, celecoxib, appeared to provide protection against radiological changes of biliary stricture, although possibly to a lesser degree than the resultant from dexamethasone. In addition, FdUrd treatment caused elevation of the DNA 2-bromo-5'deoxyuridine labeling index above control levels in biliary epithelial cells. Dexamethasone and celecoxib each significantly attenuated the FdUrd-induced elevation of DNA labeling index in biliary epithelium. These findings demonstrate the usefulness of this canine model for studying the mechanisms of drug-induced biliary sclerosis and reinforce the hypothesis that blocking inflammation may retard the progression of injury that eventually leads to fibrosis. This study suggests that clinical testing of celecoxib as a preventive for hepatic arterial-FdUrd induced biliary damage could prove valuable.

Prevention of dual promoting effects of pentachlorophenol, an environmental pollutant, on diethylnitrosamine-induced hepato- and cholangiocarcinogenesis in mice by green tea infusion.

In order to explore a possibility that the custom of drinking green tea infusion is efficacious for reducing the carcinogenic risk of environmental exposure to pentachlorophenol (PCP), we examined the effects in a hepato- and cholangiocarcinogenesis model in mice exposed to diethylnitrosamine (DEN). In the first experiment, groups of 15 male mice were initially treated with DEN at a dose of 20 p.p.m. in the drinking water for the first 8 weeks followed by a 4 week recovery interval by PCP at concentrations of 0 (basal diet), 300 or 600 p.p.m. in the diet for 23 weeks. Further groups of animals were treated with DEN and PCP in the same manner and received 2% green tea infusion (GT) instead of the drinking water from week 10 until death. PCP exposure at the high dose promoted DEN-induced hepatocarcinogenesis, and also caused progression of cystic hyperplasias of the intrahepatic bile ducts to cholangiocellular tumors. Co-administration of GT was able to prevent the increases of incidences and multiplicities of DEN-induced hepatocellular tumors and also arrest the progression of cholangiocellular tumors. In the second experiment, co-treatment with GT in the drinking water from 1 week before 300 or 600 p.p.m. PCP treatment in the diet to the end of the experiment at week 3 in B6C3F1 male mice suppressed increases of serum ALT activities, 8-oxodeoxyguanosine levels in liver DNA and bromodeoxyuridine labeling indices of hepatocytes and intrahepatic biliary epithelial cells induced by PCP. These findings suggest that regular intake of green tea may reduce the carcinogenic risk posed by an environmental pollutant, PCP, presumably due to effects on oxidative stress.

Cell Proliferation in Ovarian Carcinoma: Superior Accuracy of S-Phase Fraction (SPF) by DNA Labeling Index versus Flow Cytometric SPF, Lack of Independent Prognostic Power for SPF and DNA Ploidy, and Limited Effect of SPF on Tumor Growth Rate

Objectives. The goal of this work was to test the hypotheses that S-phase fraction (SPF) by DNA labeling index (SPF-LI) would predict the course of the disease for ovarian/peritoneal carcinomas and that SPF-LI would correlate better with pathologic classification and outcome than SPF by DNA flow cytometry (SPF-F).Methods. Tritiated thymidine (1985–1988) and bromodeoxyuridine (1988–1999) DNA labeling (SPF-LI) was evaluated in vitro on 178 tumors. Cellular DNA and SPF-F were measured flow cytometrically. During this time, 90% of ovarian/peritoneal tumors accessioned in surgical pathology were studied.Results. Tumors of low malignant potential (LMP, “borderline”) had low SPF-LI (median = 1.2%). High-grade invasive carcinomas of various types and carcinosarcomas all had high SPF-LI (medians = 11.2–23.4%). Serous low-grade invasive carcinomas (median = 1.05) resembled LMP tumors. SPF-LI of ovarian carcinomas other than LMP tumors increased slightly as FIGO stage increased (P = 0.07). Survival of patients with high-grade ovarian carcinomas was not predicted by SPF-LI or SPF-F, nor was DNA ploidy predictive. SPF-LI produced tighter distributions for various tumor types than did SPF-F. Neither SPF nor DNA ploidy contributed to prediction of outcome when tumor type and stage were included in multivariate models. We calculated the mean cell loss rate of high-grade carcinomas to be 94%.Conclusions. LMP ovarian/peritoneal tumors have low proliferation rates in contrast to high-grade carcinomas. Proliferation correlated with tumor type and stage, but neither it nor DNA ploidy predicted survival independently. Proliferation rate is growth limiting only when low. At higher levels cell loss limits growth. SPF-LI measures proliferation more accurately than SPF-F; SPF-F is not sufficiently reliable for clinical use.

Pattern of distribution of cells positive for estrogen receptor alpha and progesterone receptor in relation to proliferating cells in the mammary gland.

Since cell proliferation is indispensable for the growth and development of the breast, and estrogens are considered to play a major role in promoting cell proliferation, while progesterone influences its differentiation, the present work was designed with the purpose of verifying the relationship between cells containing steroid hormone receptors and proliferating cells in the normal human breast. Twelve breast samples were analyzed for their content of lobules type 1 (Lob1), Lob2, Lob3, and Lob4, and the number of cells containing estrogen receptor alpha (ER-alpha), progesterone receptor (PgR), or expressing Ki67 antibody was determined by double immunocytochemical technique with specific antibodies. The highest percentage of ER-alpha, PgR, and Ki67 positive cells was found in Lob1, with a progressive reduction in the more differentiated Lob2 and Lob3. ER-alpha and PgR positive cells were found exclusively in the breast epithelium and were negative for Ki67, while cells positive for Ki67 did not express receptors. These findings were compared with the distribution of ER-alpha and PgR in the autoradiographs of mammary gland of young virgin rats inoculated with 3H-thymidine for determination of the DNA labeling index (DNA-LI). Both the DNA-LI and the percentage of ER-alpha and PgR positive cells were maximal in the epithelium of terminal end buds, and these values were reduced in alveolar buds and lobules. ER-alpha and PgR positive cells did not proliferate, and those cells that had incorporated 3H-thymidine were negative for both receptors. Our results led us to conclude that the content of ER-alpha and PgR in the normal mammary tissue varies with the degree of lobular development, in parallel with cell proliferation. However, the expression of receptors occurs in cells other than the proliferating cells, indicating that they represent at least two separate cell populations. These findings open new avenues towards the understanding of the mechanisms through which estrogens and progesterone affect the proliferative activity of breast epithelial cells, and their role in the initiation of the cascade of events that leads a normal cell to cancer.

Energy metabolism and regeneration in transgenic mouse liver expressing creatine kinase after major hepatectomy

BACKGROUND & AIMS: The catalysis of a creatinine/phosphocreatine system by creatine kinase is not expressed in the liver. The aim of this study was to examine the energy energy metabolism and regeneration after hepatectomy using transgenic mouse liver expressing creatine kinase to clarify the effects of phosphocreatine on liver regeneration. METHODS: Transgenic mice were divided into two groups: group 1 was fed normal chow, and group 2 was fed 10% creatine chow for 5 days. Hepatic energy metabolism was evaluated before and after hepatectomies. Changes in remnant liver weight gain and bromodeoxyuridine labeling index were measured after 70% and 80% hepatectomies. RESULTS: Hepatic adenosine triphosphate level 24 hours after 70% hepatectomy was significantly higher in group 2 than group 1 (P<0.05). In group 2, mitochondrial adenosine triphosphate synthesis was enhanced because of elevated intramitochondrial adenine nucleotide content before hepatectomy, leading to sufficient adenosine triphosphate synthesis after a 70% hepatectomy. Bromodeoxyuridine DNA labeling index 24 hours after a 70% hepatectomy was significantly higher in group 2 than group 1. Rapid liver weight gain was observed in group 2 after a 70% hepatectomy. CONCLUSIONS: Abundant phosphocreatine promotes liver regeneration by reinforced hepatic energy metabolism. Gene transfer of creatine kinase to the liver may be a potential method in liver surgery. (Gastroenterology 1996 Apr;110(4):1166-74)

An in vitro test of the cell stretch-proliferation hypothesis of renal cyst enlargement.

In renal cystic disease, fluid accumulation within cyst lumens might stretch cyst walls and in this way stimulate cell proliferation. To test this idea, the effects of mechanical stretch on Madin-Darby canine kidney cells grown as cysts in a hydrated collagen gel or as monolayers on collagen-coated Flexcell membranes were examined. The percentage of cells synthesizing DNA (labeling index) was determined by measuring bromodeoxyuridine incorporation and counting cell numbers. The distension of single cysts for 1 h by the intraluminal injection of saline failed to produce a significant increase in labeling index. The exposure of cysts for 2.5 h to 1 mM dibutyryl cAMP + 0.1 mM isobutylmethylxanthine led to a 37% increase in luminal surface area (due to stimulated fluid secretion) and a 30% increase in labeling index. The stretch (25%) of Madin-Darby canine kidney monolayers approximately doubled the labeling index between 12 and 24 h after starting the stretch. After 48 h, the cell population density was significantly increased (P < 0.001), from 41.9 +/- 0.2 (SE; N = 12) to 48.2 +/- 0.5 (N = 12) cells/10,000 microns2. The labeling index increased linearly with applied stretch, from 7.2 +/- 0.3% (N = 36) with no stretch to 16.2 +/- 1.0% (N = 6) with 30% stretch. Stretch had to be maintained for 8 h or more to produce an increase in labeling index at 18 h. No evidence was obtained for the release of a diffusible growth factor by stretched monolayers. The increase in labeling index induced by stretch was unaffected by 50 microM gadolinium, a stretch-activated channel blocker, but was abolished by 5 micrograms/mL cytochalasin B, an actin microfilament-disrupting agent. It was concluded that prolonged stretch stimulates renal epithelial cells to synthesize DNA. This supports the idea that increased wall tension in renal cysts may stimulate cell proliferation and, thereby, may contribute to cyst enlargement.

Concerted action of TGF-beta 1 and its type II receptor in control of epidermal homeostasis in transgenic mice.

Transforming growth factor-beta 1 (TGF-beta 1) is a modulator of cellular proliferation, differentiation, and extracellular matrix deposition. It is a potent epithelial growth inhibitor and can alter the differentiative properties of keratinocytes, in vitro, but little is known about its normal physiological function in the epidermis in vivo. Transgenic mice were generated using a keratin 10 (K10) gene promoter to drive constitutive expression of TGF-beta 1 in the suprabasal keratinocyte compartment. Surprisingly, these mice showed a two- to threefold increase in epidermal DNA labeling index over control mice, in the absence of hyperplasia. The transgene, however, acted in the expected fashion, as a negative regulator of cell growth, when hyperplasia was induced by treatment by 12-tetradecanoyl-phorbol-13-acetate (TPA). Epidermal TGF-beta type I and II receptor (T beta RI and T beta RII) levels were examined in control and transgenic mice during induction of hyperplasia by TPA. Whereas T beta RI levels remained relatively constant, T beta RII expression was strongly induced in TPA-treated skins, prior to the induction of the growth inhibitory response to TGF-beta 1, and its level of expression correlated with growth sensitivity to TGF-beta 1 in vivo and in vitro. These results suggest that TGF-beta 1 and its type II receptor are part of the endogenous homeostatic regulatory machinery of the epidermis.

Effects of extracellular Ca 2+ and HCO 3− on epidermal growth factor-induced DNA synthesis in cultured rat hepatocytes

Background/Aims: The elevation of cytosolic free calcium concentration ([Ca 2+] i) and intracellular pH mediate the growth factor-initiated proliferation of many cells, but it is not known if they trigger mitosis in resting hepatocytes. The maintenance of [Ca 2+] i and intracellular pH depends partly on extracellular calcium concentration ([Ca 2+] e) and extracellular bicarbonate concentration ([HCO 3 −] e). Therefore, the effects of [Ca 2+] e and [HCO 3 −] e on hepatocyte proliferation were examined. Methods: Epidermal growth factor induced proliferation in primary cultures of rat hepatocytes. [ 3H]thymidine incorporation into DNA and nuclear labeling indices were measured. Results: Between 0.2 and 0.9 mmol/L of [Ca 2+] e, the proliferative response to epidermal growth factor increased, and total hepatocellular Ca 2+ content was increased. Increasing [HCO 3 −] e also stimulated DNA synthesis in a concentration-dependent manner, maximal at 35 mmol/L. Using optimal [Ca 2+] e (0.9 mmol/L) and [HCO 3 −] e (35 mmol/L), a synergistic stimulation of hepatocellular DNA synthesis was shown. Voltage-dependent Ca 2+ channel blockers failed to inhibit hepatocyte proliferation when administered in concentrations that inhibit proliferation in other cell types. Conclusions: [Ca 2+] e and [HCO 3 −] e are both essential for hepatocyte proliferation, and their effects are synergistic. The entry of extracellular Ca 2+ is critical for epidermal growth factor-induced DNA synthesis in hepatocytes, but this is not mediated by voltage-dependent Ca 2+ channels.

Epithelial proliferation and differentiation in the mammary gland do not correlate with cFABP gene expression during early pregnancy

Cardiac fatty acid binding protein (cFABP) is abundantly expressed in the nondividing, functionally differentiated mammary epithelium. It is very closely related, if not identical to, a previously described protein termed mammary derived growth inhibitor (MDGI). In vitro studies suggest that low concentrations of diffusible cFABP/MDGI may play a hormone-like role in limiting proliferative activity and promoting functional differentiation of this tissue, but no in vivo data to support this idea have been published. To test this hypothesis, we compared the levels of cFABP mRNA with both the epithelial DNA labelling index and levels of beta-casein mRNA in wild-type mice. We also investigated the effect of a precocious experimental increase of cFABP levels in the mammary gland of transgenic mice on the labelling index and beta-casein mRNA levels. This was accomplished by expressing a bovine cFABP cDNA under the control of the ovine beta-lactoglobulin (BLG) gene promoter. We found that although both the DNA labelling index, beta-casein mRNA levels, and cFABP mRNA levels in wild-type mice are developmentally regulated, they do not correlate with each other during early pregnancy in individual mice. Moreover, a three- to fourfold increase of total cFABP mRNA in two transgenic lines did not affect the DNA labelling index or the levels of beta-casein mRNA, an established marker of differentiation of the mammary epithelium, at this developmental stage. These data suggest that epithelial DNA synthesis, beta-casein gene expression, and expression of the cFABP gene are regulated independently in the proliferatively active mammary gland and that the rapidly dividing mammary epithelial cells are not susceptible to the action of cFABP during early pregnancy.

S-phase fraction and nuclear size in long term prognosis of patients with breast cancer.

S-phase fraction (SPF) predicts the prognosis of patients with breast cancer independently of tumor size, axillary metastasis, estrogen receptor (ER) and progesterone receptor (PgR), and patient age. Whether SPF is best measured by DNA labeling index (SPF-LI) or by flow cytometry (SPF-Flow) and what is the relative efficacy of SPF versus histopathologic characteristics for prognosis have remained unanswered questions. The authors studied 845 women with Stages I-II disease classification for years 1975-1990 with end results data, who were treated surgically with axillary lymph node dissection by an in vitro DNA labeling index protocol with tritiated thymidine or 5-bromo-2'-deoxyuridine and whose SPF was measured microscopically. Nuclear size was estimated with a calibrated optical grid as less than 11 microns, 11-14 microns, or greater than 14 microns. DNA flow cytometry was performed on fresh or paraffin embedded tissue; ER and PgR were performed by cytosol assay. Kaplan-Meier survival plots and multivariate analysis were used for comparisons. Tumor size, axillary lymph nodal status, SPF-LI, nuclear size, and ER all related strongly to breast cancer specific survival and relapse free survival. PgR was less effective. Lymph node status and tumor size predicted long term survival; differences for other variables largely disappeared by 10 years. By multivariate analysis, axillary lymph node status, tumor size, and ER were independently prognostic for disease specific, relapse free survival. A strong trend was found for nuclear size. PgR, DNA ploidy, and SPF did not contribute to prognosis independently. Nuclear size was the strongest independent predictor in patients with negative axillary lymph nodes. The number of positive axillary lymph nodes, tumor size, ER, and nuclear size were the strongest predictors of prognosis for patients with breast cancer. Only tumor size and lymph node status predicted the long term risk of metastasis.

Influence of ethanol intake on mammary gland morphology and cell proliferation in normal and carcinogen-treated rats.

Alcohol consumption has been reported to increase breast cancer risk in a majority of epidemiological studies and to enhance, at specific dietary concentrations, both the initiation and promotion stages of chemically induced rat mammary tumorigenesis. However, there is limited information regarding possible mechanisms for this effect. The present studies were conducted to examine a possible mechanism for the promoting effect of ethanol on chemically induced mammary tumorigenesis. The influence of chronic ethanol intake by female rats on the progress of differentiation and on the rate of target structure cell proliferation of the mammary gland was evaluated. Results of these studies indicate that ethanol intake at 20% and 30% of calories by female rats between the ages of 55 and 94 days (a period associated with the promotion stage of experimental mammary tumorigenesis) results in a delay in the differentiation of the mammary gland. This delay in gland maturation observed for rats consuming ethanol was evidenced by an increase in the quantity of less mature terminal-end bud (TEB) structures and a decrease in the quantity of more mature alveolar bud structures. The DNA labeling index of the target structure TEB increased significantly for rats consuming ethanol. These changes in mammary gland maturation and in TEB DNA labeling index were observed for both normal and carcinogen-treated rats consuming ethanol. Also, serum progesterone, but not estradiol, was significantly decreased for animals consuming ethanol at 30% of calories compared with isocaloric controls. The correlation of these changes in the mammary gland with the reported enhancement by ethanol of the promotion stage of experimental rat mammary tumorigenesis is discussed.

Antisense oligodeoxynucleotides targeting PDGF-B mRNA inhibit cell proliferation during embryonic rat lung development

There is increasing evidence to suggest that platelet-derived growth factor (PDGF) or PDGF-like molecules play a role in fetal lung morphogenesis. Our previous studies demonstrated the presence of PDGF-AA and PDGF-BB homodimers in embryonic and fetal rat lung. To explore further the role for PDGF-BB in embryonic lung development, we conducted intervention studies using PDGF-B chain-specific antisense oligodeoxynucleotides in a simple embryonic rat lung explant system. Unmodified antisense PDGF-B oligodeoxynucleotides inhibited, in a concentration-dependent manner, DNA synthesis of embryonic lung. A maximal inhibition of 50% was observed. The inhibitory effect of antisense PDGF-B oligodeoxynucleotides on DNA synthesis was reversed by the addition of exogenous PDGF-BB but not PDGF-AA. Antisense treatment decreased PDGF-BB but not PDGF-AA protein content, as assessed by immunoblot analyses. Incubation of lung explants with PDGF-BB neutralizing antibodies also resulted in an inhibition of DNA synthesis. Morphometric analyses of antisense-treated cultures showed a significant reduction in lung size when compared to control cultures. The epithelial component of the embryonic lungs was specifically reduced, both in mass and DNA labelling index, by antisense treatment. The number of terminal buds of the lung explants was not significantly affected by antisense PDGF-B treatment. Scrambled PDGF-B oligodeoxynucleotides had no effect. These data suggest that PDGF-BB is involved in regulating growth, but not the degree of branching, of embryonic rat lung.

Human biliary epithelial cells secrete and respond to cytokines and hepatocyte growth factors in vitro: Interleukin-6, hepatocyte growth factor and epidermal growth factor promote DNA synthesis in vitro

Recombinant growth factors and proinflammatory cytokines were added to primary cultures of human intrahepatic biliary duct epithelia to test for their ability to stimulate DNA synthesis and elicit cytokine production. Interleukin-6 and hepatocyte and epidermal growth factors were found to increase the DNA labeling index of biliary duct epithelium from fourfold to sixfold 24 hr after their addition to primary biliary duct epithelium cultures maintained in serum-free medium. The proliferative responses to all three biliary duct epithelium mitogens peaked within 24 hr, and hepatocyte growth factor was effective over a concentration range of 1.0 to 50 ng/ml, whereas interleukin-6 was effective from 1 to 1,000 U/ml. Insulin-like growth factor, phorbol myristate acetate, interleukin-1 beta and platelet-derived growth factor BB showed mild stimulatory effects, whereas interleukin-4, gamma-interferon, phytohemagglutinin and platelet-derived growth factors AA and AB did not increase DNA synthesis in biliary duct epithelium. Interleukin-1 beta and phorbol myristate acetate were also shown to induce in a dose-dependent fashion a threefold to fivefold increase of interleukin-6 production as measured by enzyme-linked immunosorbent assay in human primary biliary duct epithelium cultures, when compared with hepatocyte growth factor, epidermal growth factor, insulin-like growth factor, phytohemagglutinin, tumor necrosis factor-alpha or platelet-derived growth factor. These results show that interleukin-6 participates in growth regulation of human biliary duct epithelium. This could be exerted in a paracrine or autocrine manner.(ABSTRACT TRUNCATED AT 250 WORDS)