BrdU Labeling Index Research Articles

Increased Alveolar Concentration of Nitric Oxide Is Related to Serum-induced Lung Fibroblast Proliferation in Patients with Systemic Sclerosis

Lung inflammation is present in patients with systemic sclerosis (SSc) and interstitial lung disease (ILD), but the mechanisms linking inflammatory and fibrotic processes in ILD are unknown. Our aim was to investigate whether alveolar inflammation, reflected by increased alveolar concentration of exhaled nitric oxide (C(A)NO), is related to the ability of serum from patients with SSc to induce pulmonary fibroblast proliferation (PFP) and myofibroblast conversion. C(A)NO was measured in all subjects (37 patients with SSc and 10 healthy controls) whose sera were used to stimulate PFP (assessed by BrdU labeling index) and myofibroblast conversion (detected by alpha-smooth muscle actin expression). The PFP index in patients with SSc was compared to control values, and between patients with SSc who had elevated (> 4.3 ppb) and normal (<or= 4.3 ppb) C(A)NO values. Both C(A)NO and the PFP index were significantly greater in patients with SSc compared to controls. In patients with SSc, the PFP index was directly related to C(A)NO levels (r = 0.48; p = 0.002). The median PFP index was significantly higher in patients with SSc who had elevated C(A)NO (> 4.3 ppb; n = 25, median 1.1, range 0.98-1.23) than in patients with SSc who had normal C(A)NO (<or= 4.3 ppb; n = 12, median 0.93, range 0.82-1.08; p = 0.01). Similarly, myofibroblast conversion induced by SSc serum was significantly greater in patients with C(A)NO > 4.3 ppb than in patients whose C(A)NO was <or= 4.3 ppb (p < 0.001) and controls (p < 0.001). Alveolar inflammation reflected by increased nitric oxide production was related to serum-induced PFP and myofibroblast conversion, linking the active alveolitis process to cell proliferation and lung fibrosis in patients with SSc.

Impaired liver regeneration of steatotic rats after portal vein ligation: A particular emphasis on 99mTc-DISIDA scintigraphy and adiponectin signaling

Portal vein ligation (PVL) is increasingly employed prior to major hepatectomy in order to enhance the volume of the future liver remnant (FLR) and to avoid post-hepatectomy liver failure. The efficacy of PVL on subjects with non-alcohol fatty liver disease (NAFLD) is largely unknown. Sprague-Dawley rats fed with normal diet (control) and methionine-choline deficient diet (MCD) were used. The animals underwent PVL and were sacrificed at indicated time points. Livers from MCD rats exhibited a decreased BrdU and Ki-67 labelling index, and an increased apoptotic index after PVL compared to normal rats; as a net effect, MCD rats exhibited a decrease in their restituted liver mass and redistributed volume ratio, compared to normal rats. Normal rats displayed similar serum levels of ICG15-R before and after PVL; whereas MCD rats displayed reduced ICG15-R after PVL. Using (99m)Tc-DISIDA scintigraphy examination, livers from MCD rats exhibited decreased HEF and prolonged TE(1/2) of FLR after PVL, indicating deteriorating hepatocyte function despite the shift in volume. The basal level of plasma TNFalpha, IL-1alpha, IL-1beta, and IL-10 of MCD rats was significantly increased before PVL compared to normal rats; however their plasma level did not increase in response to PVL as in normal rats. Hepatic adiponectin mRNA surged in MCD rats after PVL, whereas its receptors, AdipoR1 and AdipoR2, were paradoxically down-regulated. PPARalpha, a down-stream molecule of AdipoR2 axis, was also decreased in MCD rats. Reduced regenerated liver mass and deteriorated hepatocyte function of the FLR from steatotic rats after PVL may be associated with deranged Kupffer cell-mediated cytokine expression and disrupted adiponectin signalling.

Effects of cold preservation and reperfusion injury on the regeneration of donor liver and associated mechanisms.

Objective To investigate the effects of cold preservation and reperfusion injury on the regen-eration of donor liver and to study the mechanisms. Methods Male SD rats were divided in to sham group (6 rats), UW 1 h group (48 rats) and UW 12 h group (48 rats). Liver tissue specimens were collected at different time points after orthotopic liver transplantation or sham operation. The morphology of liver tissue was observed via light microscope and transmission electron microscope. Proliferation of hepatocytes and sinusoidal endothelial cells (SECs) were assessed by a double immunostaining technique using antibodies against rat endothelial cell antigen-1 and bromodeoxyuridine (BrdU). Expression of vascular endothelial growth factor (VEGF) and its receptors, fins-like tyrosine kinase-1 (flt-1) and fetal liver kinase-1 (flk-1) was evaluated by immunohistochemistry. The mRNA expression of flt-1 was detected by a RT-PCR method. Mean comparison in groups was conducted by one-way ANOVA or t test. Results BrdU labeling indexes of hepatocytes and SECs in UW 12 h group was significantly higher than those in UW 1 h group (F = 61.45,41.4, P < 0.05). The proliferation of hepatocytes peaked at 48 h after operation in both UW 1 h group and UW 12 h group. However, the proliferation of SECs was fallen behind compared to hepatocytes, with a peak appeared at 72 h in UW 1 h group and at 96 h in UW 12 h group, respec-tively. The expression of VEGF was up-regulated in both UW 1 h group and UW 12 h group compared to sham group. Furthermore, expression of flt-1 and flk-1 was found to be mainly limited in SECs, with a peak in expres-sion occurring between 72 h and 96 h, coinciding with the peak in SECs proliferation in UW 1 h group. Conversely, flt-1 was found to be reduced significantly on mRNA level at any time throughout the experiment in UW 12 h group compared to sham group (F = 141.67, P < 0.05). Conclusion Reduced expression of flt-1 results in a retarded regeneration of SECs, and then the recovery of rat donor liver function is delayed after cold-preserved transplantation. Key words: Liver transplantation; Sinusoidal endothelial cells; Liver regeneration

Uterotrophic effects of cow milk in immature ovariectomized Sprague–Dawley rats

Milk contains considerable quantities of estrogens and progesterone and as such may be one of the risk factors for hormone-related cancers. To determine the hormonal effects of commercial and traditional types of milk, we performed uterotrophic tests. Forty-five rats were ovariectomized and divided into three groups of 15 animals each. The animals were kept for 7days on powdered chow and one of three different liquids: commercial milk (C), traditional milk (T), or water. At autopsy, wet and dry uterine weights were determined. The cell heights of the uterine epithelium and endometrium were determined. The uterine 5-bromo-2-deoxyuridine (BrdU) labeling index of the epithelium and endometrium gland epithelium was also assessed. The weights of wet and dry uterus were 142±13 and 112±10mg in the C group, 114±30 and 91±24mg in the T group, and 87±6 and 69±5mg in the W group. Significant differences in wet and dry uterus weights were found between all pairs of groups. The ratio of the wet uterine weight to body weight was significantly higher in the C and T groups than in the W group. The heights of the uterine epithelium and endometrium were higher and BrdU labeling index was greater in the C group than in the T and W groups. Commercially available milk and traditional milk have uterotrophic effects on young ovariectomized rats. Our findings indicate that these uterotrophic effects in the milk groups were partly due to the estrogen and progesterone in the milk.

Effect of thrombopoietin on platelet counts and liver regeneration after partial hepatectomy in a rat model

We investigated the effects of thrombopoietin (TPO) on platelet counts and liver regeneration after partial hepatectomy in rats. We performed 70% partial hepatectomy on 60 rats given either TPO or a vehicle (controls). The rats were killed 6 h, 24 h, 72 h, or 168 h after the procedure, and we examined the platelet counts, weight of the regenerated liver, hepatocyte proliferation by bromodeoxyuridine (BrdU) labeling index, and expression of hepatocyte growth factor (HGF) mRNA by reverse transcription-polymerase chain reaction. In the controls, the platelet counts were significantly lower than the basal levels, by about 20%, at 6, 24, and 72 h; then recovered to the basal level at 168 h. Conversely, in the TPO-treated rats, the platelet counts increased significantly by 17%, 35%, and 60%, at 24, 72, and 168 h, respectively. The regenerated liver regained 80% of the pre-hepatectomy weight by 72 h in the controls, whereas reconstitution of the liver was accomplished by 72 h in the TPO-treated rats. Thrombopoietin significantly enhanced the BrdU labeling index of hepatocytes and expression of HGF mRNA 24 h post hepatectomy in TPO-treated rats versus controls. Thrombopoietin increased platelet counts; thereby accelerating liver regeneration after partial hepatectomy with enhanced induction of HGF.

Specific subcellular targeting of PKCα and PKCε in normal and tumoral lactotroph cells by PMA-mitogenic stimulus

The variations of the intracellular localization of the individual protein kinase C (PKC) isoforms are related with their different biological functions. In this study, we have investigated the precise intracellular translocation of endogenous PKCalpha and PKCepsilon in PMA-stimulated normal and tumoral lactotroph cells by using confocal and immunogold electron microscopy, which was correlated with the rate of cell proliferation of both pituitary cell phenotypes. The present results showed that the short phorbol ester incubation stimulated the proliferation of normal and tumoral lactotroph cells, as determined by the measurement of the BrdU-labelling index. The translocation of PKCalpha to plasma and nuclear membranes induced by PMA was more marked than that observed for PKCepsilon in normal and tumoral lactotroph cells. Our results showed that PKCs translocation to the plasma and nuclear membranes varied from isozyme to isozyme emphasizing that PKCalpha could be related with the mitogenic stimulus exerted by phorbol ester. These data support the notion that specific PKC isozymes may exert spatially defined effects by virtue of their directed translocation to distinct intracellular sites.

Significance of Insulin Signaling in Liver Regeneration Triggered by Portal Vein Ligation

To investigate the role of insulin signaling in liver regeneration following portal vein ligation (PVL). Streptozotocin-induced insulin-deficient rats underwent PVL, and were sacrificed at indicated time points. Liver regeneration indices, including volumetric shifting, BrdU, proliferative cell nuclear antigen (PCNA), and Ki-67 labeling index, were determined. Cell cycle markers, telomerase reverse transcriptase (TERT), and apoptosis-related genes were detected using quantitative real time polymerase chain reaction (PCR). Cell cycle analysis was determined using flow cytometry. Expression of insulin-like growth factor receptor (IGFR)-2 and TGFβ1 were determined using Western blot. Restituted liver mass and redistributed volume ratio of insulin-deficient rats were decreased compared with those of normal rats. Labeling index of BrdU and PCNA of insulin-deficient rats were increased compared with normal rats, evidenced by an increased S-phase fraction detected by flow cytometry. Expression of cyclin A2, cyclin B1, TERT mRNA, and telomerase activity were decreased in insulin-deficient rats. Increased Bax, Daxx, and JNK mRNA expression and decreased Bcl X(L) expression in insulin-deficient rats, led to increased hepatocyte apoptosis than normal rats. Finally, expression of IGFR-2 was increased in insulin-deficient rats. Insulin signaling plays an important role in liver regeneration triggered by portal vein ligation.

The use of a comprehensive tumour xenograft dataset to validate gene signatures relevant for radiation response

Purpose To investigate the use of xenograft models in a novel gene signature validation method using gene expression microarrays. Materials and methods Gene expression profiles of ten human Head and Neck squamous cell carcinomas (HNSCCs) were obtained. Several published prognostic gene expression signatures were evaluated within this set. These consisted of different radiotherapy relevant signatures (i.e. for hypoxia, proliferation and ‘stemness’). Signatures were correlated with various endpoints that have been determined in the ten different xenograft models. These include immunohistochemical measures for hypoxia and proliferation, volume doubling time (VDT) and local tumour control after fractionated irradiation or after single dose irradiation under clamp hypoxia. Results We found several significant correlations between the published gene expression signatures and tumour parameters. Several signatures, like the proliferation and wound signature correlated with BrdU labelling index. Further a ‘stemness’-related gene signature showed a strong negative correlation with hypoxic fraction. Conclusions Simultaneous assessment of immunohistochemistry, in vivo tumour properties and gene expression profiling in a comprehensive set of xenograft models can be used to validate and potentially infer biological information about prognostic gene signatures.

A novel therapeutic strategy with anti-CD9 antibody in gastric cancers

CD9 is a member of the tetraspanins, and has been shown to be involved in a variety of cellular activities such as motility, cell signaling, proliferation, adhesion, and metastasis. However, very little is known about the involvement of CD9 in the process of development of primary tumors. In the present study, we investigated whether anti-CD9 monoclonal antibody (ALB6) has antitumor effects in human gastric cancer cell xenografts. Human gastric cancer cell lines (MKN-28) (5 x 10(6) cells/animal) were inoculated subcutaneously into the dorsal region of SCID mice (five mice in each group). After a tumor was visualized, animals were assigned to either the ALB6 treatment group or the control IgG treatment group (100 microg/body/time, intravenous, three times per week. Day 1, 4, and 7 of first week). Then tumor volumes were monitored every day. Proliferation of tumor was analyzed by 5-bromo-2'-deoxyuridine (BrdU) immunostaining, apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) methods, and angiogenesis was assessed by counting the number of CD34-positive endothelial cells. Tumor volume was significantly suppressed (1,682 +/- 683 mm(3) versus 4,507 +/- 1,012 mm(3); P = 0.049), the BrdU labeling indexes were significantly decreased (10.9 +/- 1.1% versus 17.2 +/- 1.4%; P = 0.009), the apoptotic indexes were significantly increased (1.98 +/- 0.48% versus 0.72 +/- 0.09%; P = 0.034), and tumor microvessel densities were significantly suppressed (671,922 +/- 34,505 pixels/mm(2) versus 1,135,043 +/- 36,086 pixels/mm(2); P = 0.037) in the ALB6 treatment group compared with the control IgG treatment group. These results suggest that administration of anti-CD9 antibody to mice bearing human gastric cancer cells successfully inhibits tumor progression via antiproliferative, proapoptotic, and antiangiogenetic effects.

Effects of co-administration of dietary sodium arsenite and an NADPH oxidase inhibitor on the rat bladder epithelium

Arsenite (As III), an inorganic arsenical, is a known human carcinogen, inducing tumors of the skin, urinary bladder and lung. It is metabolized to organic methylated arsenicals. Oxidative stress has been suggested as a mechanism for arsenic-induced carcinogenesis. Reactive oxygen species (ROS) can be important factors for carcinogenesis and tumor progression. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is known to produce intracellular ROS, therefore, we investigated the ability of apocynin (acetovanillone), an NADPH oxidase inhibitor, to inhibit the cytotoxicity and regenerative cell proliferation of arsenic in vitro and in vivo. Apocynin had similar effects in reducing the cytotoxicity of As III and dimethylarsinous acid (DMA III) in rat urothelial cells in vitro. When tested at the same concentrations as apocynin, other antioxidants, such as l-ascorbate and N-acetylcysteine, did not inhibit As III-induced cytotoxicity but they were more effective at inhibiting DMA III-induced cytotoxicity compared with apocynin. In vivo, female rats were treated for 3 weeks with 100 ppm As III. Immunohistochemical staining for 8-hydroxy-2′-deoxyguanosine (8-OHdG) showed that apocynin reduced oxidative stress partially induced by As III treatment on rat urothelium, and significantly reduced the cytotoxicity of superficial cells detected by scanning electron microscopy (SEM). However, based on the incidence of simple hyperplasia and the bromodeoxyuridine (BrdU) labeling index, apocynin did not inhibit As III-induced urothelial cell proliferation. These data suggest that the NADPH oxidase inhibitor, apocynin, may have the ability to partially inhibit arsenic-induced oxidative stress and cytotoxicity of the rat bladder epithelium in vitro and in vivo. However, apocynin did not inhibit the regenerative cell proliferation induced by arsenite in a short-term study.

Protocol to assess growth plate kinetics during long‐term in vivo multiphoton imaging using BrdU as a physiological marker

With the ability to collect optical images deep into biological tissues, multiphoton microscopy (MPM) is a powerful tool to study growth plate kinetics in real time, but the act of imaging introduces variables with the potential to alter chondrocytic kinetics. This study is to develop a procedure to assess chondrocytic viability during in vivo MPM imaging on the proximal tibial growth plate of 4‐wk‐old mice. The BrdU labeling index is used as a physiological marker to assess imaging variables, following IACUC procedures. Isoflurane anesthesia was used at 1L/min (no ventilation) to maintain breathing at ~1.5/sec. Fluorescein was injected IP for a vascular concentration of ~1mM. After 2 hrs, a second dye injection was given with ~200 ul of isotonic fluids to avoid hydropenia. Body temperature was maintained at ~350C. The surgical limb was bathed in warm Ringer's. BrdU was given IP 0.5 hr before euthanasia. Fixation and plastic embedment followed, with analysis of BrdU LI by standard protocols. Maintenance of core and limb temperature, hydration, reinjection of the dye after 2 hrs, and limiting imaging to 3.5 hrs proved to be key to successful long‐term imaging. This standard protocol for BrdU‐based analysis of proliferative capacity will allow comparative assessment of multiple vital dyes for monitoring chondrocytic dynamics during multi‐hour MPM imaging. Supported by NIH‐R01AR052003.Grant Funding SourceNIH

Feasibility of Reduced-size Orthotopic Liver Transplantation with Fatty Grafts and its Potential Regeneration in Rats

We undertook this study to determine the minimum necessary volume of various grades of fatty liver grafts in reduced-size orthotopic liver transplantation (ROLT) and to elucidate the property of fatty liver regeneration. By modified two-cuff technique, ROLT of different rats was performed with various grades and volumes of fatty liver grafts. The survival rate, graft-to-recipient body weight ratio (GRWR), standard liver volume (SLV) and liver function were detected to determine appropriate steatotic grafts that could be safely adopted in ROLT. In grade II macrovesicular steatosis (MaS) grafts, the 1-week survival rate of 70% ROLT was significantly worse (25%). In grafts with microvesicular steatosis (MiS) undergoing 70%, 60%, and 50% ROLT, 7-day survival decreased according to the graft volume (75%, 75%, and 33.3%). Proliferating cell nuclear antigen (PCNA) and 5-bromo-2'-deoxyuridine (BrdU) labeling index in normal liver graft reached the highest on postoperative day (POD) 3, whereas in fatty grafts the expression peak was on POD 7. Liver regeneration ratios of normal liver graft were higher than mild MaS graft, but grade II MaS grafts hardly regenerated. When compared with moderate MaS grafts B2 (70% ROLT) and mild MaS grafts A3 (60%) groups, the disparities of alanine aminotransferase and total bilirubin were significant. In successful and long-term survival of rat ROLT, MiS or grade II MaS livers could be safely used as grafts only when their GRWR >2.29+/-0.20 (or 2.28+/-0.12) % and SLV >60%; MaS livers in grade II should not be used as grafts. Hepatic regeneration potential of mild MaS or MiS with any grade graft is not obviously impaired after ROLT, but delayed proliferative peak and prolonged cell proliferative cycle are shown.

Effect of estrogen on the blood supply of pituitary autografts in rats

Estrogens are known to cause pituitary enlargement and lactotroph proliferation. They also modulate pituitary angiogenesis and induce tumor formation. Pituitary grafts, due to the loss of hypothalamic dopamine, also show lactotroph hyperplasia. We investigated the role of estrogen on rat pituitary autograft vascularization by light and transmission electron microscopy, and assessed prolactin (PRL) blood levels, microvessel density (MVD) and cell proliferation using the BrdU labeling index. All adenohypophysial cell types were identified by immunohistochemistry (streptavidin-biotin-peroxidase complex method). The proangiogenic factors, vascular endothelial growth factor (VEGF), its receptor Flk-1, and hypoxia inducible factor-1alpha (HIF-1alpha) were similarly demonstrated. The prevalence of lactotrophs, as well as more intense staining for VEGF, Flk-1 and HIF-1alpha, was noted in those grafts exposed to estrogen, mainly in the area surrounding the central necrotic core. Immunostaining showed Flk-1 expression increased in endothelial cells of the estrogen-exposed grafts as compared with those unexposed. In contrast to the grafts not exposed to estrogen, in the estrogen-exposed grafts, only fenestrated endothelium could be demonstrated, suggesting that estrogen induces fenestration of newly formed capillaries. There was an increase in blood PRL levels in the estrogen-treated groups as compared with controls. Both MVD and BrdU labeling indices were higher in grafts exposed to estrogen, especially after 4 weeks. Our results suggest that estrogen administration not only enhances the expression of proangiogenic factors in the pituitary grafts but also induces their expression at earlier stages, leading to rapid neoformation of purely fenestrated capillaries.

Prolonged oestrogen treatment does not correlate with a sustained increase in anterior pituitary mitotic index in ovariectomized Wistar rats.

Oestrogen is a powerful mitogen that is believed to exert a continuous, dose-dependent trophic stimulus at the anterior pituitary. This persistent mitotic effect contrasts with corticosterone and testosterone, changes in the levels of which induce only transient, self-limiting fluctuations in pituitary mitotic activity. To further define the putative long-term trophic effects of oestrogen, we have accurately analysed the effects of 7 and 28 days oestrogen treatment on anterior pituitary mitotic activity in ovariectomized 10-week-old Wistar rats using both bromodeoxyuridine (BrdU) and timed colchicine-induced mitotic arrest. An oestrogen dose-dependent increase in mitotic index was seen 7 days after the start of treatment as expected, representing an acceleration in gross mitotic activity from 1·7%/day in ovariectomized animals in the absence of any oestrogen replacement to 3·7%/day in the presence of a pharmacological dose of oestrogen (50 mcg/rat per day: ∼230 mcg/kg per day). Despite continued exposure to high-dose oestrogen and persistence of the increase in pituitary wet weight, the increase in mitotic index was unexpectedly not sustained. After 28 days of high-dose oestrogen treatment, anterior pituitary mitotic index and BrdU-labelling index were not significantly different from baseline. Although a powerful pituitary mitogen in the short term, responsible, presumably, for increased trophic variability in oestrus cycling females, these data indicate that in keeping with other trophic stimuli to the pituitary and in contrast to a much established dogma, the mitotic response to longer-term high-dose oestrogen exposure is transient and is not the driver of persistent pituitary growth, at least in female Wistar rats.

Host Susceptibility to Schistosomes: Effect of Host Sera on Cell Proliferation of Schistosoma mansoni Schistosomula

To determine effects of the sera on cell proliferation, schistosomula of Schistosoma mansoni (20-days-old) were incubated in medium containing fetal calf serum plus hamster (highly susceptible host) portal or peripheral venous serum, or rat (poorly susceptible host) portal or peripheral venous serum in the presence of bromodeoxyuridine (BrdU). Compared with schistosomula cultured in presence of control medium containing fetal calf serum alone, BrdU labeling indices (BLIs) were increased by 39% in the presence of portal, but not in peripheral, serum of hamsters. In contrast, no significant differences were observed in the BLIs in rat portal, or peripheral, sera or in control media. In vivo BrdU labeling results revealed that there was no detectable cell proliferation in S. mansoni schistosomula (6 days old) in the lungs. However, cell proliferation was detected in schistosomula beginning at 17 days. The results indicated that portal venous serum from a highly susceptible host, but not from a poorly susceptible host, stimulated schistosome cell proliferation in vitro. The timing of the increase in cell proliferation in terms of development corresponded to liver portal-mesenteric localization of schistosomula. Together, the data support the conclusion that in susceptible hosts, portal serum may play a role in schistosome cell proliferation, possibly resulting in termination of schistosome migration. This may explain the colocalization of adults, and the known organ selectivity of disease.

Molecular Indicators of High-Risk Disease.

Abstract High-risk multiple myeloma (HRMM) is routinely defined by laboratory parameters alone or in combination in the Durie-Salmon and, more recently, the ISS staging systems. The Bartl grade, a cell morphology-based staging system, has seen limited use. The presence of abnormal cytogenetics, high BrdU labeling index, interphase FISH abnormalities, and flow cytometric measures have also been used. A molecular-based classification and risk stratification of MM may improve the definition of HRMM. Global gene expression profiling (GEP) with of CD138-selected plasma cells followed by unsupervised hierarchical cluster analysis revealed that MM comprises a spectrum of seven distinct reproducible subtypes. A validated molecular classification schema has been defined as follows: (MS = t(4;14); MF = t(14;16) or t(14;20); CD-1 = t(11;14) or t(6;14) and CD-2 = t(11;14) or t(6;14) with high CD20 and/or VPREB3), hyperdiploidy (HY = high DKK1, FRZB, NCAM1, TNFSF10), low bone disease (LB = NF-kB signature, high CCND2, CST6, and IL6R) and proliferation (PR = high MIK67, CCNB1, CCNB2, TOP2A, and TYMS). Correlating GEP with outcome in two independent cohorts permitted the identification of a high-risk signature (UAMS 17-gene model), present in approximately 13% of newly diagnosed disease. GEP and high-resolution comparative genomic hybridization in 92 cases confirmed that the altered expression of the 17 genes in the model is driven by 1q gains and 1p losses. This high-risk signature is evident in a subset of all 7 molecular subtypes and negatively influences outcome. For example, low-risk MS disease fares much better than high-risk MS disease. We recently reported that the addition of bortezomib to TT3 has significantly improved outcome in low-risk MS disease, thereby demonstrating the value of GEP in evaluating benefits of new treatments that might be otherwise masked. When subjected to multivariate analysis including the International Staging System (ISS) and a gene expression-based proliferation index (GEP PI), the UAMS 17-gene model remained a significant predictor of outcome. Mulligan and colleagues developed outcome classifiers for relapsed disease treated with single agent bortezomib or high dose dexamethasone improved upon the risk stratification provided by the ISS. These predictive models showed some specificity for bortezomib. Using U133A data from newly diagnosed disease treated with ASCT, the Mayo clinic group validated the UAMS 17-gene model, but also showed that the t(4;14) translocation remained a significant adverse variable. The IFM recently reported on a 15-gene model of high-risk (IFM 15-gene model) related to cell proliferation. Multivariate showed that the UAMS 17-gene model was significant in all datasets, while the IFM 15-model was significant in a limited number. This difference might be attributed to the dependence of the IFM model to cell proliferation. GEP on 71 paired diagnostic and relapse samples indicate that the UAMS 17-gene model score increases in 80% of the cases and a low-risk to high-risk conversion in 14 of 24 (58%) severely impacted post-relapse survival. Expression of TP53 is a surrogate for 17p13 deletion, and TP53 expression below a specific threshold (seen in approximately 10% of newly diagnosed disease) imparts a poor prognosis in low-risk – but not high-risk – MM, defined by the UAMS 17-gene model. In conclusion, while the majority of patients with MM can anticipate long-term disease control, approximately 25% of patients with molecularly defined HRMM do not benefit from current approaches.

Enhancement of preneoplastic lesion yield by Chios Mastic Gum in a rat liver medium-term carcinogenesis bioassay

The mastic ( Pistacia lentiscus var. chia) tree is native throughout the Mediterranean region and has long proved a source of food additives and medical treatments. To investigate the modifying effects of Chios Mastic Gum on rat liver carcinogenesis, 6-week-old male F344 rats were subjected to the established rat liver medium-term carcinogenesis bioassay (Ito-test). At the commencement, rats (groups 1–4) were intraperitoneally injected with 200 mg/kg body weight of diethylnitrosamine (DEN). After two weeks, mastic was added to CRF (Charles River Formula)-1 powdered basal diet at doses of 0, 0.01, 0.1 and 1% in groups 1–4, respectively. At week 3, all rats were underwent two-thirds partial hepatectomy. The experiment was terminated at week 8. As results show, liver weights were significantly increased in a mastic dose-dependent manner among groups 1–4. The numbers (/cm 2) and the areas (mm 2/cm 2) of glutathione S-transferase placental form (GST-P)-positive cell foci (≥ 0.2 mm in diameter) were significantly increased in the DEN-1% group compared to the DEN-alone group, along with the average areas per foci and larger-sized foci (≥ 0.4 mm). 5-Bromo-2′-deoxyuridine (BrdU) + GST-P double-immunohistochemistry showed the highest BrdU-labeling indices within GST-P foci in the DEN-1% group. 8-hydroxydeoxyguanosine (8-OHdG) levels in liver DNA did not vary, while real-time quantitative polymerase chain reaction (PCR) analysis of livers revealed many up- or down-regulated genes in the DEN-1% group. In conclusion, this is the first report to display a promotion potential of Chios Mastic Gum on the formation of preneoplastic lesions in the established rat liver medium-term carcinogenesis bioassay.

Inhibitory Effect of Curcumin on Early Liver Regeneration Following Partial Hepatectomy in Rats

Curcumin (Cur) is a nontoxic, hepatoprotective antioxidant. Recent investigations have demonstrated a protective effect of curcumin pretreatment during cold ischemia of hepatocytes, but its impact on liver regeneration per se has not been investigated so far. Male Sprague-Dawley rats (n = 6 per group) underwent sham operation, 70% partial hepatectomy (PH), or PH with curcumin application (100 mg per kg bodyweight per day) starting 48 h before surgery. Rats were sacrificed 24 h after surgery. Liver regeneration was analyzed by measurement of relative liver weight, mitotic-index, bromo-deoxy-uridine (BrdU)-incorporation and Ki-67 expression. The relative liver weight 24 h after surgery was similar in the PH groups with and without curcumin treatment. Also, a comparably high number of Ki-67 positive proliferating hepatocytes was detected in both groups. In contrast, the mitotic index in the untreated PH group (83 +/- 20 mitosis/2000 hepatocytes) was significantly higher than in the curcumin treated group (21 +/- 6). The BrdU labeling index was slightly higher in the curcumin treated group with PH (24% +/- 5%) than in the untreated group (16% +/- 2%). The hepatocyte density as marker of cellular hypertrophy was significantly lower in the curcumin group (474 +/- 23) than in the untreated group (609 +/- 22). Curcumin inhibits cell cycle progression during normal liver regeneration in rats, predominantly at the level of the G2/M transition point. However, the total liver mass and function was not significantly altered. Nevertheless, application of curcumin in conditions of high physiological cell proliferation should be performed with caution.

Furan-induced dose–response relationships for liver cytotoxicity, cell proliferation, and tumorigenicity (furan-induced liver tumorigenicity)

Rodent studies of furan are associated with liver cell necrosis, release of liver-associated enzymes, increased hepatocyte proliferation, and hepatocarcinogenesis. For carcinogens whose proposed mode of action is cytolethality, it is hypothesized that the dose–response curve for tumor development would parallel the dose–response curve for cell death with compensatory proliferation in the target organ. To prospectively test this hypothesis, female B6C3F 1 mice were exposed to furan at carcinogenic doses and lower for 3 weeks or 2 years. At 3 weeks and in the 2-year study, there were dose-dependent and significant increases in hepatic cytotoxicity at 1.0, 2.0, 4.0, and 8.0 mg furan/kg. For cell proliferation as measured by 5-bromo-2′-deoxyuridine (BrdU) labeling index (LI), there was a statistically significant trend with increasing dose levels of furan and increased LI at 8.0 mg/kg. There was an increased incidence of foci of altered hepatocytes, hepatocellular adenomas, and adenomas or carcinomas at 4.0 and 8.0 mg/kg and carcinomas at 8.0 mg/kg. The multiplicity of microscopic tumors was increased and latency was decreased in mice exposed to 8.0 mg/kg. Prevalence of hepatic nodules at necropsy was increased in mice exposed to 4.0 and 8.0 mg/kg. Data demonstrate an association among furan-induced hepatic cytotoxicity, compensatory cell replication, and liver tumor formation in mice; at high doses ⩾4.0 mg/kg, furan induced hepatotoxicity, compensatory cell replication and tumorigenesis in a dose-related manner, while furan did not produce tumors at cytotoxic doses of 1.0 and 2.0 mg/kg.

Relationship between GST Yp Induction and Hepatocyte Proliferation in Rats Treated with Phase II Drug Metabolizing Enzyme Inducers

Butylated hydroxyanisole (BHA) and 1,2-bis(2-pyridyl)ethylene (2PY-e) are phase II drug metabolizing enzyme inducers which cause hepatomegaly without hepatocyte hypertrophy and induce glutathione S-transferase Yp (GST Yp, pi-class GST), which is known as a tumor marker. To evaluate the relationship between GST Yp induction and hepatocyte proliferation, male F344/DuCrj rats were treated with BHA, 2PY-e, or phenobarbital (PB) for three or seven days. All three chemicals caused increases in liver weight after three and seven days. Immunohistochemical examinations revealed that BHA and 2PY-e induced GST Yp in the hepatocytes of the periportal and centrilobular areas at three and seven days, respectively, whereas PB did not. Significant increases in the BrdU labeling indices were found in the livers of rats in each of the three-day treatment groups, but the labeling index of rat livers treated with BHA was decreased to the control level at seven days, although the high labeling indices of 2PY-e and PB persisted at seven days. Double immunostaining confirmed that BrdU-positive nuclei corresponded to GST Yp-positive hepatocytes in both BHA and 2PY-e treated rats. These results suggest that the GST Yp induction caused by BHA or 2PY-e has some kind of relationship with hepatocyte proliferation.