Proliferating Cellular Nuclear Antigen Research Articles

Characterization of GFAP expression and cell proliferation in the rat median eminence following hypophysectomy.

To analyze whether the reorganization of the rat median eminence after hypophysectomy might be related to changes in glial fibrillary acidic protein (GFAP)- and cellular proliferation, the distribution of cells immunoreactive for GFAP and the proliferation rate of such cells were analyzed at 20, 40 and 60 days posthypophysectomy. For this study, four rostro-caudal regions of the median eminence were differentiated: the retrochiasmatic, preinfundibular, infundibular and postinfundibular regions. In each of these regions, three layers were studied: the ependymal, the internal and the external. At 20 and 40 days after hypophysectomy, significant increases in cellular proliferation affecting all three layers studied in the preinfundibular and infundibular regions were found. At the same time points, increases in GFAP expression were also observed. However, after 60 days, GFAP and proliferative cellular nuclear antigen (PCNA) expression decreased. Although variations of PCNA and GFAP levels were evident, no colocalisation of PCNA and GFAP was found in the cells of the median eminence in untreated or hypophysectomized rats when sections were analyzed by double immunohistochemical staining. Our results suggest that reorganization of median eminence involves alterations (or modulation) of GFAP-immunoreactive cells together with a proliferation of cells that are not GFAP-immunoreactive. This study also demonstrates that this reorganization is completed within the first two months after hypophysectomy.

Genetic markers of survival and liver recurrence after resection of liver metastases from colorectal cancer.

A significant number of patients with liver metastases from colorectal cancer (CRC) achieve 5-year survival after liver resection. Increased expression of genetic markers in the primary tumor are known to predict outcome after colonic resection, but the predictive value of such markers after resection of hepatic metastases is unknown. The objective of this study was to evaluate whether DNA content and multiple genetic markers, separately or expressed together, can predict patient outcome (liver recurrence and survival) after resection of hepatic metastases. We studied the paraffin-embedded liver tissue of 71 consecutive patients who had undergone a potentially curative resection of hepatic metastases from CRC. Using DNA flow cytometry and immunohistochemical staining techniques we determined the DNA content and the level of co-expression of seven tumor-associated proteins: proliferating cellular nuclear antigen (PCNA), epidermal growth factor receptor (EGFr), p53, c-erbB-2, H-ras, c-myc, and nm23. Three endpoints (liver recurrence, cancer specific, overall survival) were correlated with these tumor markers. The 5-year overall survival of the group was 31.2%. There was no correlation detected between the DNA aneuploidy and overall or cancer-specific survival. Similarly, expression of the individual tumor-associated proteins did not predict survival. Patients whose tumors co-expressed multiple markers had survivals similar to those whose tumors expressed fewer markers. However, a significant difference in hepatic recurrence was found between the p53-positive and p53-negative patients (p = 0.007), with marker-negative tumors having decreased recurrence. In conclusion, this study demonstrates that the DNA content and genetic markers c-myc, c-erbB-2, EGFr, H-ras, p53, PCNA, and nm23 do not predict survival after potentially curative resection of hepatic metastases from CRC. However, the immunoreactivity of p53 may be an important marker of local recurrence in the liver, which may be useful if re-resection of metastatic liver tumors is considered a viable management option in this disease.

External-beam radiation therapy for improved dialysis access patency: feasibility and early safety.

Rectenwald, J. E., Pretus, H. A., Seeger, J. M., Huber, T. S., Mendenhall, N. P., Zlotecki, R. A., Palta, J. R., Li, Z. F., Hook, S. Y., Sarac, T. P., Welborn, M. B., Klingman, N. V., Abouhamze, Z. S. and Ozaki, C. K. External-Beam Radiation Therapy for Improved Dialysis Access Patency: Feasibility and Early Safety. Radiat. Res. 156, 53-60 (2001).Prosthetic dialysis access grafts fail secondary to neointimal hyperplasia at the venous anastomosis. We hypothesized that postoperative single-fraction external-beam radiation therapy to the venous anastomosis of hemodialysis grafts can be used safely in an effort to improve access patency. Dogs (n = 8) underwent placement of expanded polytetrafluoroethylene grafts from the right carotid artery to the left jugular vein. Five dogs received single-fraction external-beam photon irradiation (8 Gy) to the venous anastomosis after surgery. Controls were not irradiated. Shunt angiograms were completed 3 and 6 months postoperatively. Anastomoses, mid-graft, and the surrounding tissues were analyzed. Immunohistochemistry for smooth muscle cell alpha-actin, proliferating cellular nuclear antigen (PCNA), and apoptosis was performed. Incisions healed well, though all animals developed wound seromas. One control suffered graft thrombosis 4 months postoperatively. Angiography/histology confirmed severe neointimal hyperplasia at the venous anastomosis. The remaining seven dogs developed similar amounts of neointimal hyperplasia. PCNA studies showed no accelerated fibroproliferative response at irradiated anastomoses compared to controls. Skin incisions and soft tissues over irradiated anastomoses revealed no radiation-induced changes or increase in apoptosis. Thus we conclude that postoperative single-fraction external-beam irradiation of the venous anastomosis of a prosthetic arteriovenous graft that mimics the situation in humans is feasible and safe with regard to early wound healing.

Expression of osteopontin in gentamicin-induced acute tubular necrosis and its recovery process

There is controversy regarding the exact localization and roles of osteopontin (OPN), a multipotential chemokine, in renal injury. There is little information on the expression and role of OPN in gentamicin-induced acute tubular necrosis (ATN) and its recovery process. A severe ATN model was made using male Wistar rats by injecting gentamicin (150 mg/kg/day) for five days and limiting the provision of water. The expression and localization of OPN mRNA and protein, ED1 as a macrophage marker, proliferating cellular nuclear antigen (PCNA), CD44 as an OPN receptor, megalin as a proximal tubule marker, and their relationships to each other were examined from the early tubular necrotic period to the late recovery period by Northern blotting, in situ hybridization, and double immunohistochemical staining. In the gentamicin group, OPN mRNA and protein were expressed in only the PCNA-positive proliferating cortical distal tubules, not in the necrotic proximal tubules, until day 6 after the first administration, but were found markedly in PCNA-positive regenerative proximal and distal tubules on days 10, 15, and 30. The localization of PCNA-positive cells was almost always accompanied with the up-regulated expression of OPN using quantitative analysis (P < 0.01). CD44 expression was markedly up-regulated in the renal cortical tubular epithelium from days 6 to 30. In the control group, no expression of OPN and CD44 in the cortical area was found throughout the experimental period. These results suggested that OPN is related to the proliferation and regeneration of tubular epithelial cells after tubular damage.

Blasts from elderly acute myeloid leukemia patients are characterized by low levels of culture- and drug-induced apoptosis

Increasing evidence suggests that the biology of acute myeloid leukemia (AML) may differ between older and younger patients, with a higher incidence of antecedent myelodysplasia, unfavorable cytogenetic abnormalities, and multidrug resistance seen in the elderly. Abrogation of apoptosis in response to cytotoxic medications is associated with drug resistance in AML, as is expression of bcl-2, an important anti-apoptotic protein. We hypothesized that blasts from elderly (≥55 years) and young adult AML patients might have different levels of apoptotic and cell cycle responses to chemotherapeutic agents, as well as different levels of proliferation and of bcl-2 protein expression. Therefore, we cultured bone marrow leukemia samples from previously untreated elderly ( n=33) and young ( n=21) AML patients for 48 h and then measured apoptosis, bcl-2 protein levels, cell cycle distributions, and expression of a proliferation marker, proliferating cellular nuclear antigen (PCNA) in multi-parametric flow cytometry assays. In some experiments, leukemia samples were exposed to cytarabine (Ara-C) or daunomycin (DNR) for the last 16–18 h of the culture period. In comparison to samples from young patients, cultured samples from elderly AML patients had a higher fraction of viable cells, as measured by Trypan blue exclusion, higher PCNA expression, and significantly less culture-induced and drug-induced apoptosis. The mean apoptosis after culture was 13% for elderly AML samples, versus 20% for young AML samples ( P=0.009). Similarly, the mean apoptosis after Ara-C was lower in elderly than in young AML samples, 13 versus 28% ( P=0.001), as was the mean apoptosis after DNR, 15 versus 26% ( P=0.012). Diminished apoptotic responses in elderly AML cells were not consistently associated with high bcl-2 levels at thaw or bcl-2 levels increased by culture. These data suggest that new therapies should be developed to overcome abrogated apoptosis, particularly in elderly AML patients.

Effects of physiological versus pharmacological beta-carotene supplementation on cell proliferation and histopathological changes in the lungs of cigarette smoke-exposed ferrets.

There remains a remarkable discordance between the results of observational epidemiological studies and intervention trials using beta-carotene as a potential chemopreventive agent. One question that needs to be examined is whether the adverse outcomes of human beta-carotene trials are related to the large doses of beta-carotene that were administered. In the present study, ferrets were given a physiological (low) dose or a pharmacological (high) dose of beta-carotene supplementation (0.43 mg versus 2.4 mg/kg body wt/day, which is equivalent to 6 mg versus 30 mg/day in humans) and exposed to cigarette smoke for 6 months. We investigated the effects of these doses of beta-carotene on retinoid concentrations, expression of retinoic acid receptors (RARs), activator protein 1 (AP-1; c-Jun and c-Fos), cyclin D1, proliferating cellular nuclear antigen (PCNA), and histopathological changes in the lungs of both normal and cigarette smoke-exposed ferrets. Thirty-six male ferrets were treated in six groups-control, smoke-exposed (SM), low-dose beta-carotene (LBC), high-dose beta-carotene (HBC), low-dose beta-carotene plus smoke exposure (LBC+SM) or high-dose beta-carotene plus smoke exposure (HBC+SM)-for 6 months. Retinoic acid concentration and RAR beta gene expression, but not expression of RAR alpha and RAR gamma, was reduced in the lung tissue of HBC+SM, HBC, SM and LBC+SM ferrets, but not in that of LBC ferrets, as compared with the control group. Expression of AP-1 and PCNA was greater in HBC+SM, HBC, SM and LBC+SM ferrets, but not in the LBC ferrets, as compared with the control group. Increased amounts of cyclin D1 and keratinized squamous metaplasia were observed in the lung tissue of HBC+SM, HBC and SM groups but not in that of the LBC+SM, LBC or control groups. These data suggest that, in contrast with a pharmacological dose of beta-carotene, a physiological dose of beta-carotene in smoke-exposed ferrets has no potentially detrimental effects and may afford weak protection against lung damage induced by cigarette smoke.

Neogenesis of pancreatic endocrine cells in copper-deprived rat models.

Transplantation of progenitor cells for regeneration of islet cells could prove invaluable in the treatment of diabetes mellitus. This study provides evidence that in rats maintained on a copper-deficient diet containing the copper-chelating agent tetraethylenepentamine pentahydrochloride, regeneration of single alpha and beta endocrine cells in the ductules and acinar tissue of the adult rat pancreata occurred. These regenerated cells both in the ductules and acinar tissue stained positive for glucagon and insulin similar to cells within the islets and in addition to being reactive to proliferative cellular nuclear antigen, an intracellular marker of active proliferation. In contrast, the control group pancreata did not show any evidence of islet regeneration, proliferation, or proliferative cellular nuclear antigen reactivity pre- or posttransplantation. Transplantation of digested pancreatic tissues from the copper-deficient group into the spleen of syngeneic diabetic rats reversed diabetes, and this was confirmed histologically by demonstrating cells within ductules that stained positively for insulin. This study concludes that copper deprivation contributes to the neogenesis of pancreatic alpha and beta cells in the ductules and acinar tissue of adult pancreas in rat model and that transplanted stem cells maintain their functional capacity in the recipient after transplantation.

Acute carbon tetrachloride feeding selectively damages large, but not small, cholangiocytes from normal rat liver.

The aim of this study was to develop a model of selective duct damage restricted to hormone-responsive segments corresponding to the ducts damaged in primary biliary cirrhosis (PBC). Carbon tetrachloride (CCl4) was fed by gavage to rats, and 2, 7, 14, and 28 days later, small and large cholangiocytes were isolated. Apoptosis was determined in situ by morphology and in purified cholangiocytes by assessment of nuclear fragmentation by 4, 6-diamidino-2-phenylindole (DAPI) staining. Cholangiocyte proliferation was evaluated in situ by morphometry of liver sections stained for cytokeratin-19 (CK-19) and by proliferating cellular nuclear antigen (PCNA) staining in liver sections and in purified cholangiocytes by PCNA gene expression. Ductal secretion was assessed by measurement of secretin receptor (SR) gene expression and secretin-induced cyclic adenosine 3',5'-monophosphate (cAMP) synthesis and secretin-induced choleresis. Two days after CCl4 administration, there was an increased number of small ducts, but a reduction of large ducts. Apoptosis, observed only in large ducts, was associated with decreased DNA synthesis and ductal secretion. Conversely, small cholangiocytes expressed de novo the SR gene and secretin-stimulated cAMP synthesis 2 days after CCl4 treatment. Proliferation of large cholangiocytes was delayed until 7 days, which was associated with a transient increase in ductal secretion in vivo. CCl4 effects on cholangiocytes were reversed by day 28. CCl4 treatment causes a decrease in large duct mass as a result of a higher rate of apoptosis and absence of initial proliferation in large cholangiocytes. These processes were concomitant with a decrease of ductal secretion in large cholangiocytes. Small cholangiocytes appear resistant to CCl4-induced apoptosis, and proliferate and transiently compensate for loss of proliferative and secretory activity of large cholangiocytes.

Expression of polo-like kinase (PLK) in the mouse placenta and ovary.

The polo-like kinase (PLK) is a mammalian serine/threonine kinase involved in cell cycle regulation. Much evidence for the role of PLK in the cell cycle has come from studies of cultured cells; however, little is known about its function or even expression in vivo. The present study examined the features of PLK expression in the mouse placenta and ovary. Immunohistochemical studies showed that PLK is highly expressed in the basement membrane of the endometrial gland, in some endothelial cells, in endometrium after embryo implantation, in trophoblastic tissue invading the decidua, in the ovarian stroma and in some lutein bodies. In contrast, PLK was not detectable by immunohistochemistry in endometrial stroma before decidualization, in decidua, in trophoblastic tissue not invading the decidua or in ovarian follicles. PLK expression seemed to be correlated with the expression of proliferation cellular nuclear antigen (PCNA) in many placental and ovarian cells, reflecting a role in cellular proliferation. Nevertheless, in ovarian stroma and lutein bodies where PCNA was not expressed, PLK was strongly expressed. This finding indicates that PLK may have some post mitotic functions in certain specialized cell types.

The role of calcium-sensing receptor abnormality in the pathogenesis of secondary hyperparathyroidism.

because the nodular lesion is at an advanced stage of Introduction parathyroid hyperplasia. In order to elucidate the relationship of CaR and 2In uraemic secondary hyperparathyroidism (2-HPT), HPT more precisely, the expression of CaR mRNA a normal serum calcium (Ca) does not adequately and protein was studied in surgically resected parasuppress excessive parathyroid hormone (PTH) prothyroid glands of six patients with severe 2-HPT (intact duction and secretion [1]. This result suggests an PTH>800 pg/ml ) on haemodialysis. Light microscopy abnormal Ca-sensing mechanism in 2-HPT. The sensitshowed that all glands included hypertrophic and ivity of the parathyroid to extracellular Ca has been hyperplastic lesions with or without nodular change. evaluated by the sigmoidal curve between serum intact Immunohistological examination demonstrated the PTH and Ca concentrations [2]. Although there is decreased expression of CaR protein in all hyperplastic controversy that the set-point Ca concentration calcuparathyroid glands tissues, in particular in nodular lated from this curve precisely reflects the sensitivity hyperplastic lesions compared with diffuse lesions. In of the parathyroid cell for Ca [3–5], the depressed addition, the expression of CaR mRNA by quantitative sensitivity for Ca has been clearly shown in vitro and RT–PCR and Northern blotting analyses showed a by the common experience in severe 2-HPT that high PTH develops despite the unphysiological hypercalcaemore profound reduction in large nodular hyperplastic mia. The mechanism for this abnormality has been parathyroid glands compared to that in small hyperplpartially explained by decreased plasma-activated vitastic parathyroid glands. The finding that the expresamin D, an increased parathyroid cellular mass, or a sion of parathyroid gland CaR mRNA and protein reduced vitamin D receptor in the parathyroid gland. decreases particularly in large nodular hyperplastic However, the precise mechanism is not yet fully glands is consistent with the results of two previous understood. reports. We next evaluated the correlation between resected gland weight and the expression of CaR mRNA, which Ca-sensing receptor in 2-HPT was classified into four grades by mRNA density. A significant inverse correlation was observed between Since the discovery of the Ca-sensing receptor (CaR) the weight and CaR mRNA of resected parathyroid in bovine [6 ] and human parathyroid [7], much attenglands. tion has been directed at the relationship between CaR Parathyroid cells in nodular hyperplasia generally and abnormal sensitivity for Ca in 2-HPT. Kifor et al. show a greater proliferative change compared with first reported the decreased expression of CaR protein those in diffuse hyperplasia [11]. Therefore, the relation in parathyroid tissue [8]. This finding was also supbetween proliferative activity and CaR protein expresported by CaR mRNA levels [9]. sion in the parathyroid cell was examined. Proliferation These two reports together demonstrate that the was determined by proliferative cellular nuclear antigen downregulation of CaR is more remarkable in nodular (PCNA) staining. CaR protein was strongly expressed hyperplastic lesions of the parathyroid compared with in parathyroid tissue where the PCNA staining was that in diffuse lesions and that the same downregulation scarce. In contrast, CaR expression was rare in tissue is observed in the hyperplastic tissue of primary hyperwhere the PCNA was strongly stained. These results parathyroidism. Since hyperplastic parathyroid glands suggest that the proliferative potential of parathyroid display less sensitivity to Ca [10], it appears to be tissue inversely correlates with CaR expression and reasonable that CaR expression and the sensitivity for sensitivity for Ca. Ca are suppressed more in nodular hyperplastic glands, The relation between cell proliferation and expression of CaR is similar to that between cell proliferation Correspondence and offprint requests to: Tadao Akizawa, 1-30 Fujigaoka, Aoba-ku, Yokohama, 227, Japan. and vitamin D receptor density [12]. Expression of

Cardiac remodelling in experimental renal failure--an immunohistochemical study.

In view of the high rate of cardiac death in renal failure, the factors involved in the genesis of structural changes in the heart are of obvious interest. The present study addresses the issue whether growth factors known to be involved in cardiac remodelling are abnormally expressed in rats with renal failure. Sprague Dawley rats were subjected to binephrectomy (2 days) or subtotal nephrectomy (8 weeks). Controls were sham-operated rats and rats with Goldblatt hypertension. Cardiac expression of proliferating cellular nuclear antigen (PCNA), of growth factors and of their receptors (PDGF, TGF-beta, VEGF) was investigated immunohistochemically. In addition, cardiac PDGF-and TGF-beta mRNA were assessed using quantitative RT-PCR. Eight weeks after subtotal nephrectomy (SNX) significantly increased expression of PCNA and PDGF was found in the cardiac interstitium and of PCNA and VEGF in the walls of intramyocardial arteries. In addition, PCNA-positive cardiomyocytes were noted in SNX. Similar changes were not seen in the hearts of hypertensive controls, i.e. rats with renovascular hypertension, despite slightly higher blood pressure and more pronounced left ventricular hypertrophy (LVH). While significant changes of cardiac PDGF- and TGF-beta mRNA expression could not be documented in the whole-heart homogenates 8 weeks after subtotal nephrectomy, 2 days after bilateral nephrectomy PDGF mRNA was significantly increased and TGF-beta mRNA decreased. The observations demonstrate (i) specific activation of cardiac interstitial cells after SNX, (ii) activation of postmitotic cardiomyocytes, possibly predisposing to apoptosis, (iii) increased expression of PDGF in the cardiac interstitium and in the wall of intramyocardial arteries, (iv) increased expression of VEGF associated with hypertrophy of arterial smooth muscle cells. These results were not explained by elevated blood pressure or LVH, respectively.

Expression of Id-1 mRNA and Protein in the Post-Ischemic Regenerating Rat Kidney

The basic helix-loop-helix (bHLH) class of proteins are of major importance in controlling tissue-specific gene expression. The actions of the bHLH proteins are inhibited by a related class of proteins, inhibitors of differentiation (Id). We have studied the expression of one of these latter proteins, Id-1, in the normal and post-ischemic regenerating rat kidney by immunocytochemistry, Western blot and RNase protection assay (RPA) and correlated Id-1 regulation to the expression of vimentin and proliferating cellular nuclear antigen (PCNA). In the normal kidney strong immunostaining for Id-1 was found in the distal nephron, especially in the distal convoluted tubule in the cortex. In particular, the perinuclear region was intensely stained in the cells of the distal tubule. mRNA for Id-1 was detectable by RPA on total RNA extracted from the renal cortex of sham-operated animals. The Id-1 monomer was detected on Western blots of normal animals. Vimentin was expressed in the mesangial cells of the glomeruli and in cells in the interstitium while tubule cells were negative. The labeling intensity for PCNA was low in all cellular compartments in the normal kidney. In the regenerating kidneys at various time intervals, the expression of Id-1-like immunoreactivity was widespread in the regenerating dedifferentiated tubule cells while by the end of the study period, more highly differentiated tubule cells appeared to lose their staining. On Western blots the Id-1 monomer was undetectable and instead strong staining was seen in the high molecular range. Id-1 mRNA levels in the regenerating kidneys did not differ significantly when compared to sham. PCNA labeling was intense in the regenerating kidneys at all time periods studied, indicating the intense proliferative activity in the regenerating kidneys. Vimentin expression in the renal tubule cells was increased from day 3 and onward. The data are consistent with a hypothesis in which Id-1 regulates differentiation of renal tubule cells in the post-ischemic regenerating rat kidney.

Significance of Cyclin D1 Overexpression in Transitional Cell Carcinomas of the Urinary Bladder and its Correlation With Histopathologic Features

BACKGROUND. Genetic alterations leading to neoplastic transformation of the uroChyi Chia R. Lee, M.D. thelium are likely to involve the activation of oncogenes and loss of functional Shinji Yamamoto, M.D. tumor suppressor genes. Cyclin D1 has been implicated as a putative protooncoKeiichirou Morimura, M.D. gene whereas mutations of the p53 gene occur frequently in invasive transitional Hideki Wanibuchi, M.D. cell carcinomas (TCCs) of the urinary bladder. In this study, cyclin D1 overexNobuyasu Nishisaka, M.D. pression and nuclear accumulation of p53 were evaluated and the results correlated Shinichi Ikemoto, M.D. with histopathologic features. Tatsuya Nakatani, M.D. METHODS. TCCs of the urinary bladder from 161 surgical procedures were evaluSeiji Wada, M.D. ated for cyclin D1 overexpression and nuclear accumulation of p53. Results were Taketoshi Kishimoto, M.D. correlated with tumor grade, T classification, and papillary status. Topologic distriShoji Fukushima, M.D. butions of cyclin D1, p53, and proliferating cellular nuclear antigen (PCNA) were evaluated. Northern blot analysis was performed on selected specimens. 1 First Department of Pathology, Osaka City UniRESULTS. Overexpression of cyclin D1 was observed in 47% (24 of 51) of Grade 1 versity Medical School, Osaka, Japan. TCCs and 20% (13 of 65) of Grade 2 TCCs but in no Grade 3 TCCs. Approximately 34% (14 of 41) of Ta classified TCCs and 21% (13 of 63) of T1 classified TCCs 2 Department of Urology, Osaka City University Medical School, Osaka, Japan. were immunoreactive for cyclin D1 whereas none of the TCCs beyond T1 was immunoreactive. Overexpression of cyclin D1 was observed only in papillary type TCCs. Results of Northern blot analysis for cyclin D1 were comparable to those of immunohistochemistry. CONCLUSIONS. The observed significant relation between cyclin D1 overexpression and tumor grade/T classification suggests that cyclin D1 may be a useful biologic marker for biopsied materials or urine cytology specimens. The prognostic significance of cyclin D1 overexpression in TCCs remains to be determined. Cancer 1997; 79:780–9. q 1997 American Cancer Society.

Significance of cyclin D1 overexpression in transitional cell carcinomas of the urinary bladder and its correlation with histopathologic features.

Genetic alterations leading to neoplastic transformation of the urothelium are likely to involve the activation of oncogenes and loss of functional tumor suppressor genes. Cyclin D1 has been implicated as a putative protooncogene whereas mutations of the p53 gene occur frequently in invasive transitional cell carcinomas (TCCs) of the urinary bladder. In this study, cyclin D1 overexpression and nuclear accumulation of p53 were evaluated and the results correlated with histopathologic features. TCCs of the urinary bladder from 161 surgical procedures were evaluated for cyclin D1 overexpression and nuclear accumulation of p53. Results were correlated with tumor grade, T classification, and papillary status. Topologic distributions of cyclin D1, p53, and proliferating cellular nuclear antigen (PCNA) were evaluated. Northern blot analysis was performed on selected specimens. Overexpression of cyclin D1 was observed in 47% (24 of 51) of Grade 1 TCCs and 20% (13 of 65) of Grade 2 TCCs but in no Grade 3 TCCs. Approximately 34% (14 of 41) of Ta classified TCCs and 21% (13 of 63) of T1 classified TCCs were immunoreactive for cyclin D1 whereas none of the TCCs beyond T1 was immunoreactive. Overexpression of cyclin D1 was observed only in papillary type TCCs. Results of Northern blot analysis for cyclin D1 were comparable to those of immunohistochemistry. The observed significant relation between cyclin D1 overexpression and tumor grade/T classification suggests that cyclin D1 may be a useful biologic marker for biopsied materials or urine cytology specimens. The prognostic significance of cyclin D1 overexpression in TCCs remains to be determined.

Changes in Biomarker Expression in the Development of Prostatic Adenocarcinoma

In spite of the prevalence of prostatic adenocarcinoma, the development and natural history of this malignancy is poorly understood. This paper reviews the current knowledge of biomarker expression during the development and progression of prostatic adenocarcinoma. Emphasis is placed on the comparison of biomarker expression in benign prostatic epithelium, intraepithelial neoplasia (PIN), a putative preinvasive lesion, and prostatic adenocarcinoma. Within the benign epithelium, the proliferative potential is restricted to the basal cells as demonstrated by the expression of proliferating cellular nuclear antigen (PCNA). The strong expression of the bcl-2 protein, an inhibitor of apoptosis, supports the concept that the basal cells or a subpopulation of the basal cells represent the stem cell of the epithelium. In addition, the strong expression of growth factor receptors such as the epidermal growth factor receptor (EGFr), p185erbB-2, p180erbB-3, and c-met suggests that the growth of the basal cells is regulated by autocrine or paracrine factors. The luminal cells express secretory products such as prostate specific antigen and prostatic acid phosphatase, but demonstrate little expression of PCNA as well as growth factor receptors and proto-oncogene products. These observations are consistent with the theory that the luminal cell population is derived from the differentiation of the basal cells. In contrast to the normal epithelium, PCNA expression is frequently detected in the dysplastic luminal cells of the PIN lesion. Likewise, strong expression of p185erbB-2, p180erbB-3 and the c-met proto-oncogene product is also detected in the luminal cells of PIN lesions. Other factors which are strongly expressed by the dysplastic luminal cells include the nm23-Hl gene product, tumor associated glycoprotein-72 (TAG-72), fatty acid synthetase (FASE) and proteolytic enzymes. These findings suggest that PIN lesions are derived from an impairment of the differentiation of basal cells. The majority of biomarkers such as PCNA, p185erbB-2, p180erbB-3, TAG-72, nm23-Hl and FASE which are strongly expressed in PIN lesions are also expressed in prostatic adenocarcinoma supporting the concept that PIN is a preinvasive lesion. Mutations of the p53 tumor suppressor gene, as well as strong expression of transforming growth factor a and bcl-2 typically occur in advanced stage prostatic adenocarcinomas and therefore likely represent late events in the development of prostatic adenocarcinoma.

Transforming growth factor beta 1 induces mitogenesis in fetal rat brown adipocytes.

The presence of transforming growth factor beta 1 (TGF-beta 1) for 24 or 48 h stimulated DNA synthesis, the percentage of cells in the S + G2/M phases of the cell cycle, and cell number, as compared to quiescent cells. The mitogenic capacity of TGF-beta 1 (1 pM) was similar to that shown by 10% fetal calf serum (FCS). TGF-beta 1 for 48 h increased by 5-fold the percentage of cells containing (3H)thymidine-labeled nuclei as compared to quiescent cels. In addition, single fetal brown adipocytes, showing their typical multilocular fat droplets phenotype, become positive for (3H)thymidine-labeled nuclei in response to TGF-beta 1. Moreover, TGF-beta 1 induced the mRNA expression of a complete set of proliferation-related genes, such as c-fos (30 min), c-myc and beta-actin (2 h), and H-ras, cdc2 kinase, and glucose 6-phosphate dehydrogenase (G6PD) at 4 and 8 h, as compared to quiescent cells. Concurrently, TGF-beta 1 for 12 h increased the protein content of proliferating cellular nuclear antigen (PCNA) by 6-fold and p21-ras by 2-fold. Although our results demonstrate that TGF-beta 1 induces the expression of very early genes related to cell proliferation, TGF-beta 1 could be acting either as a mitogen or as a survival factor in induce proliferation to fetal brown adipocytes.

Low affinity NGF receptor expression in CMT1A nerve biopsies of different disease stages.

Duplication of the gene for the peripheral myelin protein 22 (PMP22) is the most common cause for Charcot-Marie-Tooth neuropathy type 1a (CMT1A) neuropathy. In early stages of the disease PMP22 is overexpressed in nerve biopsies from CMT1A patients. Recent studies with genetically modified Schwann cells have demonstrated that the altered expression of PMP22 modulates cell growth. Thus we hypothesized that elevated expression of PMP22 at the beginning of the disease might alter Schwann cell differentiation and phenotype. In this study we investigated Schwann cell phenotype in different stages of CMT1A neuropathy using antibodies to established Schwann cell markers. We found a pathological expression of low affinity nerve growth factor receptor (LNGF-R-also referred to in the literature as p75) in numerous Schwann cells of young CMT1A patients with almost normal myelin thickness and very few onion bulbs. During further progression of the disease, when Schwann cells began to form onion bulbs, we observed an intense LNGF-R immunoreactivity in all layers of the onion bulbs. In the most advanced stages of the disease, characterized by massive onion bulb formation, no LNGF-R immunoreactivity was shown. In age-matched control nerves LNGF-R staining was barely detectable. Furthermore, onion bulbs seen in patients with chronic idiopathic polyneuropathy (CIDP) were always negative for LNGF-R. In addition, at all CMT1A disease stages analysed, LNGF-R-positive Schwann cells were glial fibrillary acidic protein (GFAP) negative. Immunostaining with an antibody to the proliferation marker, proliferating cellular nuclear antigen (PCNA) indicated Schwann cell proliferation when onion bulb formation was well developed. In conclusion, we describe a disease stage-dependent altered Schwann cell phenotype in CMT1A neuropathy, which could be a direct consequence of the PMP22 overexpression on Schwann cell growth behaviour or, less likely, a secondary phenomenon related to myelin loss.

Modulation of salt transport rate affects DNA synthesis in vivo in rat renal tubules

In adult male Wistar rats we investigated whether cell proliferation contributes to salt load-induced hypertrophy of distal tubules. In one treatment group salt transport in the thick ascending limb (TAL) was inhibited by furosemide (7.5 mg/100 g body wt/24 hr, via osmotic minipump) and stimulated in the successive distal segments by simultaneous high salt intake (F + Salt). Controls without furosemide treatment had a standard salt intake. All animals received the thymidine analog bromodeoxyuridine (BrdU) during 24 and 72 hours, respectively. In cryostat sections of the perfusion-fixed kidneys DNA synthesis was assessed by immunohistochemistry for BrdU, and for endogenous proliferating cellular nuclear antigen (PCNA). Incidence of BrdU- and PCNA-labeled nuclei were quantified in proximal tubules, medullary TAL, and cortical distal segments downstream the TAL. In control animals low labeling indices were found in all investigated segments. After 24 and 72 hours of F + Salt, indices of labeled nuclei were markedly increased in distal segments downstream the TAL, whereas they were significantly reduced in TAL. In proximal tubules increased DNA synthesis rate was apparent only after 72 hours. The data demonstrate that (1.) DNA synthesis rate in nephron segments in vivo varies in parallel with changes of their salt transport activity; (2.) increased DNA synthesis, thus probably cellular proliferation, is a component of the structural response of nephron segments following increased salt transport activity.

Analysis of proliferative grade in glial neoplasms using antibodies to the Ki-67 defined antigen and PCNA in formalin fixed, deparaffinized tissues

In an effort to optimize immunocytochemical methods to evaluate cell kinetics in brain tumors, we studied two newly-developed antibodies which react with formalin resistant epitopes of Proliferating Cellular Nuclear Antigen (PCNA) and Ki-67. These results were compared with standard flow cytometric cell cycle data from the same tumor specimens to determine if these methods correlate with each other, and whether retrospective analysis using these antibodies is feasible for cell kinetic analysis of brain tumors. Thirty-one specimens of glial tumors submitted for flow cytometry during 1992 were also reacted with antibodies to PCNA (PC-10) and Ki-67 (MIB-1). Flow cytometry scores for S-phase Fraction were compared with immunocytochemical scores for both antibodies, using an arbitrary rating of 1 (low, < 4%), 2 (intermediate, 4-6%), 3 (high, > 6%), and 1 (< 25% positive), 2 (26-75% positive), 3 (> 75% positive), respectively. MIB-1 results were found to correlate significantly with the S-phase fraction as determined by flow cytometry. The MIB-1 data showed a trend toward underestimating, i.e., lower scores, the proliferative index compared with flow cytometry. There was less of a correlation between PC-10 antibody scores and flow cytometry S-phase fraction, as PC-10 immunostaining typically overestimated the proliferative rate of brain tumors when compared with flow cytometry. There was an exact correlation between PC-10 and MIB-1 in only 4 cases, whereas in the remaining specimens, PC-10 results were always higher than MIB-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Le(y) antigen expression is correlated with apoptosis (programmed cell death).

Apoptosis (programmed cell death) is a basic physiological process which determines specific patterns of tissue size and shape, and balance of cell number, during morphogenesis, and seems to play an integral role in oncogenic progression. Since dramatic changes of cellular glycosylation pattern are well known to be closely correlated with differentiation, development and oncogenesis, it is likely that similar specific changes are associated with apoptosis. However, this possibility has not been systematically investigated. We therefore carried out histological studies of many tumours and normal tissues for which a high incidence of apoptosis is believed to occur. Sections were stained with monoclonal antibodies (MoAbs) directed to carbohydrate antigens Le(y) and Le(x), proliferating cellular nuclear antigen (PCNA) and Fas (previously claimed to be an apoptosis-inducing antigen). Antibody staining patterns were compared with morphological cell characteristics as revealed by haematoxylin/eosin staining, and DNA fragmentation patterns (a marker of apoptosis) as revealed by 3'-OH nick-end labelling technique. We found that expression of Le(y) (defined by MoAb BM1) is closely correlated with the process of apoptosis, but not with cell proliferation or necrosis. Within Le(y)-positive areas of tissue sections, typical apoptotic morphological changes and DNA fragmentation (as revealed by positive nick-end labelling) were frequently observed in certain loci, although not all Le(y)-positive cells showed such signs of apoptosis. Le(y)-positive areas showed consistent negative staining by MoAb directed to PCNA and negative or weak staining by MoAb directed to Fas antigen, regardless of tissue source. No such trends were observed for Le(x) glycosylation. We conclude that Le(y) expression is a useful phenotypic marker predictive of apoptosis, i.e. some (although not all) Le(y)-positive cells subsequently become apoptotic.