BrdUrd Labeling Index Research Articles

Histopathologic validation of 3′-deoxy-3′-18F-fluorothymidine PET for detecting tumor repopulation during fractionated radiotherapy of human FaDu squamous cell carcinoma in nude mice18F-FLT PET repopulation -->

Background and purposeFaDu human squamous cell carcinoma (FaDu-hSCC) demonstrates accelerated tumor repopulation during fractionated irradiation with pathological validation (Ki-67 and BrdUrd makers) in a xenograft model system. However, these and other functional assays must be performed ex vivo and post hoc. We propose a novel, in vivo, real-time assay utilizing 18F-FLT PET. Material and methodsNude mice with FaDu-hSCC were irradiated with 12 or 18 fractions of 1.8Gy ([Dm]=3.0Gy), either daily or every second day. 18F-FLT micro-PET scans were performed at different time points, FLT parameters (SUVmax, SUVmean, and T/NT) were measured. Tumor sections were stained for Ki-67 and BrdUrd, a labeling index (LI) was calculated. Imaging-pathology correlation was determined by comparing FLT parameters and immunohistochemical results. ResultsMeasured SUVmax, SUVmean and T/NT decreased significantly after daily irradiation with 12 fractions in 12days (P<0.05) and 18 fractions in 18days (P<0.05). In contrast, these parameters increased in mice treated with 12 fractions in 24days (P>0.05) and 18 fractions in 36days (P>0.05), suggesting accelerated repopulation. Similarly, Ki-67 and BrdUrd LIs demonstrated significant decreases with daily irradiation (P<0.05), and increases with every-second-day irradiation (P>0.05). 18F-FLT parameters correlated strongly with proliferation markers (r2: 0.679–0.879, P<0.001). Conclusions18F-FLT parameters were in good agreement with Ki-67 and BrdUrd Li. These results may support a potential role for 18F-FLT PET in real-time detection of tumor repopulation during fractionated radiotherapy.

Effect of celecoxib on inhibiting tumor repopulation during radiotherapy in human FaDu squamous cell carcinoma.

Aim of the studyFaDu human squamous cell carcinoma (FaDu-hSCC) demonstrated accelerated tumor repopulation during fractionated irradiation with pathological validation in a xenograft model system. Previous studies showed that the selective cyclooxygenase (COX)-2 inhibitor celecoxib can enhance the tumor response to radiotherapy. So we aimed to explore the effect of celecoxib in inducing apoptosis and inhibiting repopulation of FaDu tumors in nude mice during fractionated radiotherapy.Material and methodsFaDu-hSCC was transplanted into the right hind leg of BALB/C nude mice. Mice were treated with celecoxib and/or fractionated irradiation. Celecoxib (100 mg/kg/day) was administered by daily gavage. Irradiation was delivered with 12 to 18 fractions of 3.0 Gy daily or every second day based on Petersen's repopulation model. At different time points, tumors were excised for immunohistochemistry staining.ResultsSignificant tumor repopulation occurred after about 18 days of radiotherapy. On average, Ki-67 and bromodeoxyuridine (BrdUrd) labeling indices (LI) decreased with daily irradiation (both p < 0.05) and increased with every-second-day irradiation (both p > 0.05), suggesting accelerated repopulation. Ki-67 LI decreased in celecoxib concurrent with radiotherapy for 12 fractions in 24 days and 18 fractions in 36 days compared with irradiated alone (p = 0.004 and 0.042, respectively). BrdUrd LI values were lower in the concurrent groups than irradiated alone (p = 0.001 and 0.006, respectively). Epithelial growth factor receptor (EGFR) expression score decreased in the concurrent groups than irradiated alone (p = 0.037 and 0.031, respectively). Caspase-3 expression scores were higher in the concurrent groups than irradiated alone (p = 0.05 and 0.006, respectively).ConclusionsCelecoxib concurrent radiotherapy could inhibit tumor repopulation and increase tumor apoptosis during the treatment in FaDu squamous cell carcinoma.

Effects of an early lipopolysaccharide challenge on growth and small intestinal structure and function of broiler chickens

Hu, X. F., Guo, Y. M., Li, J. H., Yan, G. L., Bun, S. and Huang, B. Y. 2011. Effects of an early lipopolysaccharide challenge on growth and small intestinal structure and function of broiler chickens. Can. J. Anim. Sci. 91: 379–384. Two experiments were conducted to determine the effect of early exposure to lipopolysaccharide (LPS) on small intestinal structure and function of broiler chickens. Seven-day-old birds were randomly allotted to two equal treatments: an LPS-injected treatment in which the birds were injected intraperitoneally with LPS 500 µg kg−1 body weight (dissolved in 1 mL saline) on 8, 10, 12, 15, 17, and 19 d of age, i.e., on days 1, 3, and 5 d for 2 continuous weeks, and a control treatment (CTRL) in which the birds were similarly injected with 1 mL saline as a placebo. In exp. 1, food intake and weight gain were monitored over the 2 wk, the weight of the small bowel was determined at 14 and 21 d of age and duodenal and jejunal villus height and crypt depth, D-xylose uptake were also measured at 21 d. In exp. 2, additional measurements of the intestinal peristalsis ratio and the BrdU-labeling index and duodenal sodium-glucose co-transporter-1 (SGLT1) mRNA level were made at 21 d of age. The results showed that LPS challenge decreased feed intake, daily gain, duodenal and jejunal villus height and crypt depth, plasma D-xylose concentration and intestinal BrdUrd-labeling index, respectively (P&lt;0.05) as well as small bowel weight at 14 and 21 d of age (P&lt;0.05). Conversely, LPS injection increased SGLT1 mRNA level in the small intestine (P&lt;0.05) and the small intestinal relative weight at 14 (P&lt;0.05) and 21 d of age (P=0.063). Following LPS injection there were non-significant changes in feed conversion ratio and intestinal peristalsis ratio (P&gt;0.05). In conclusion, early LPS challenge delayed the growth of intestine and impaired small intestinal structure and absorptive function.

Corticosterone Administration Alters Small Intestinal Morphology and Function of Broiler Chickens

Two experiments were carried out to study the effects of corticosterone (CORT) administration on intestinal morphology and function of broilers. In both experiments, birds were randomly divided into two equal groups. One group was the control group (CTRL), and the birds were fed with a basal diet. The other was the experimental group (CORT), and the birds were fed with the basal diet plus 30 mg of CORT/kg diet. At 21 days of age, performance, morphological characteristics of intestine, D-xylose level in plasma, activities of digestive enzymes in digesta, digestibility of nutrients and 5-bromo-2-deoxyuridine (BrdUrd)-labeling index of intestinal epithelial cells were determined. CORT administration decreased feed intake, daily gain and feed conversion ratio (p 0.05). CORT administration increased activities of trypsin and amylase (p<0.05), and decreased BrdUrd-labeling index of duodenal and jejunal epithelial cells (p<0.05). In conclusion, CORT administration impaired the normal morphology and absorptive capacity of the small intestine of broiler chickens.

Critical Role for Transcription Coactivator Peroxisome Proliferator-activated Receptor (PPAR)-binding Protein/TRAP220 in Liver Regeneration and PPARα Ligand-induced Liver Tumor Development

Disruption of the gene encoding for the transcription coactivator peroxisome proliferator-activated receptor (PPAR)-binding protein (PBP/TRAP220/DRIP205/Med1) in the mouse results in embryonic lethality. Here, we have reported that targeted disruption of the Pbp/Pparbp gene in hepatocytes (Pbp(DeltaLiv)) impairs liver regeneration with low survival after partial hepatectomy. Analysis of cell cycle progression suggests a defective exit from quiescence, reduced BrdUrd incorporation, and diminished entry into G(2)/M phase in Pbp(DeltaLiv) hepatocytes after partial hepatectomy. Pbp(DeltaLiv) hepatocytes failed to respond to hepatocyte growth factor/scatter factor, implying that hepatic PBP deficiency affects c-met signaling. Pbp gene disruption also abolishes primary mitogen-induced liver cell proliferative response. Striking abrogation of CCl(4)-induced hepatocellular proliferation and hepatotoxicity occurred in Pbp(DeltaLiv) mice pretreated with phenobarbital due to lack of expression of xenobiotic metabolizing enzymes necessary for CCl(4) activation. Pbp(DeltaLiv) mice, chronically exposed to Wy-14,643, a PPARalpha ligand, revealed a striking proliferative response and clonal expansion of a few Pbp(fl/fl) hepatocytes that escaped Cre-mediated gene deletion in Pbp(DeltaLiv) livers, but no proliferative expansion of PBP null hepatocytes was observed. In these Pbp(DeltaLiv) mice, none of the Wy-14,643-induced hepatic adenomas and hepatocellular carcinomas was derived from PBP(DeltaLiv) hepatocytes; all liver tumors developing in Pbp(DeltaLiv) mice maintained non-recombinant Pbp alleles and retained PBP expression. These studies provide direct evidence in support of a critical role of PBP/TRAP220 in liver regeneration, induction of hepatotoxicity, and hepatocarcinogenesis.

Age and Bromodeoxyuridine Labelling Index as Prognostic Factors in High-grade Gliomas Treated with Surgery and Radiotherapy

Aims To determine the prognostic value of proliferative potential and DNA ploidy in 72 brain tumours (36 grade III and 36 grade IV astrocytomas) using bromodeoxyuridine (BrdUrd) incorporation and flow cytometry. Material and methods All 72 patients underwent excision, mostly incomplete of the tumour. After surgery, eight patients received conventionally fractionated radiotherapy, 11 patients received accelerated radiotherapy, and 53 patients received hypofractionated radiotherapy. Tumour samples taken during surgery from each patient were incubated in vitro for 1 h at 37°C with BrdUrd using the high pressure oxygen method. The percentage of BrdUrd-labelled cells (BrdUrd labelling index [BrdUrd LI]), and the total DNA content were evaluated. Results The tumours showed variability in the BrdUrd LI values, which ranged from 0.3 to 19.1%. No difference was observed in mean BrdUrd LI between grade III and grade IV sub-groups. A significantly higher percentage of DNA aneuploidy was observed in grade III gliomas (69.4%) than in grade IV gliomas (52.8%). Univariate analysis showed that younger patients (≤51 years) ( P = 0.021) with grade III gliomas ( P = 0.030) and low tumour proliferation rate (BrdUrd LI ≤ 2.7%, P = 0.028) had significantly higher 5-year survival rates. Tumour ploidy had no influence on patients' survival ( P = 0.591). However, Cox multi-variate analysis showed that only age over 51 years, and high tumour proliferation rate (BrdUrd LI > 2.7%), were significant unfavourable prognostic factors in patient survival. Conclusion In this study, independent prognostic factors for patients with high-grade gliomas treated with surgery and post-operative radiotherapy are age and tumour proliferation rate assessed according to the BrdUrd LI.

Dynamics of Hypoxia, Proliferation and Apoptosis after Irradiation in a Murine Tumor Model

Proliferation and hypoxia affect the efficacy of radiotherapy, but radiation by itself also affects the tumor microenvironment. The purpose of this study was to analyze temporal and spatial changes in hypoxia, proliferation and apoptosis after irradiation (20 Gy) in cells of a murine adenocarcinoma tumor line (C38). The hypoxia marker pimonidazole was injected 1 h before irradiation to label cells that were hypoxic at the time of irradiation. The second hypoxia marker, CCI-103F, and the proliferation marker BrdUrd were given at 4, 8 and 28 h after irradiation. Apoptosis was detected by means of activated caspase 3 staining. After immunohistochemical staining, the tumor sections were scanned and analyzed with a semiautomatic image analysis system. The hypoxic fraction decreased from 22% in unirradiated tumors to 8% at both 8 h and 28 h after treatment (P < 0.01). Radiation did not significantly affect the fraction of perfused vessels, which was 95% in unirradiated tumors and 90% after treatment. At 8 h after irradiation, minimum values for the BrdUrd labeling index (LI) and maximum levels of apoptosis were detected. At 28 h after treatment, the BrdUrd labeling and density of apoptotic cells had returned to pretreatment levels. At this time, the cell density had decreased to 55% of the initial value and a proportion of the cells that were hypoxic at the time of irradiation (pimonidazole-stained) were proliferating (BrdUrd-labeled). These data indicate an increase in tumor oxygenation after irradiation. In addition, a decreased tumor cell density without a significant change in tumor blood perfusion (Hoechst labeling) was observed. Therefore, it is likely that in this tumor model the decrease in tumor cell hypoxia was caused by reduced oxygen consumption.

Prognostic significance of proliferation rate and DNA ploidy in astrocytic gliomas treated with radiotherapy

Summary Aim The proliferative potential, and DNA ploidy in 50 brain tumours (15 grade I & II, and 35 grade III & IV astrocytomas) were investigated using bromodeoxyuridine (BrdUrd) incorporation and flow cytometry. Materials/Methods Tumour samples taken from each patient during surgery were incubated in vitro for one hour at 37°C with bromodeoxyuridine (BrdUrd), using the high pressure oxygen method. The percentage of BrdUrd-labelled cells (BrdUrd Labelling index, BrdUrd LI), and the total DNA content were evaluated. After surgery, 21 patients received conventionally fractionated radiotherapy (RT), 11 patients received accelerated RT, and 18 patients underwent hypofractionated RT. Results The tumours showed variability in BrdUrd LI values, which ranged from 0.3 to 15.8%. A significantly higher mean value for BrdUrd LI was shown in grades AIII & IV (3.5%), than in astrocytomas of grades AI & II (1.5%, p=0.005). A lower though not statistically significant percentage of DNA aneuploidy was observed in low-grade (40.2%) glioma than was seen in high-grade (65.7%) glioma. Univariate analysis showed that younger (≤50 years) patients (p=0.001), those with AI & II glioma (p=0.000), low tumour proliferation rate (BrdUrd LI ≤2.1%, p=0.006) and conventional or hypofractionated RT (p=0.000) had a significantly higher 5-year survival rate. Tumour ploidy had no influence on patients’ survival (p=0.261). However, a Cox multivariate analysis showed that only the patients’ age (>50 years), high grade tumours (AIII & IV) and accelerated RT were significantly unfavourable prognostic factors in terms of survival. Conclusions To improve RT results, younger patients (≤50 years) with fast proliferating tumours should receive more aggressive treatment.

Effects of nicotinamide and carbogen in different murine colon carcinomas: Immunohistochemical analysis of vascular architecture and microenvironmental parameters

Purpose To investigate oxygenation, perfusion, and cell proliferation in two murine colon carcinoma lines with known differences in chemotherapy sensitivity and analyze the effect of nicotinamide and carbogen on these tumor characteristics. Methods and materials Mice with s.c. transplanted C38 and C26a murine colon tumors were treated with nicotinamide and carbogen and compared with control tumors. Two markers of hypoxia, CCI-103F and pimonidazole, were injected before and after treatment with nicotinamide/carbogen, respectively, allowing each tumor to serve as its own control. Hoechst33342 was used as a perfusion marker and bromodeoxyuridine (BrdUrd) as a proliferation marker. Frozen tumors were cut for multistep immunostaining and computer-controlled microscope scanning for hypoxic fractions (HF), perfused fractions (PF), vascular density, and BrdUrd-labeling index (LI). Results Microscopic observation of C38 and C26a tumors showed extensive differences in vascular architecture, distribution patterns of hypoxia, and BrdUrd-labeling. Quantitative analysis of C38 and C26a tumors showed a decrease in HF in response to all treatment modalities. For C38 tumors, the average decrease in HF in response to carbogen containing treatments was larger than to nicotinamide alone. In C26a tumors, no difference in average decrease in HF was observed between the treatments. The PF of C38 and C26a did not change in response to treatment. The LI of C38 and C26a decreased upon all treatments, which was statistically significant in the combination treatment of C38. Conclusions The mechanism that can simultaneously explain all the observed changes in response to treatment may be the conversion of metabolism from less respiration toward more glycolysis due to increased glucose levels (Crabtree effect), although other mechanisms of actions cannot be excluded.

Increased Susceptibility of Mice Lacking Clara Cell 10-kDa Protein to Lung Tumorigenesis by 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone, a Potent Carcinogen in Cigarette Smoke

Ninety percent of all human lung cancers are related to cigarette smoking. Both tobacco smoke and lung tumorigenesis are associated with drastically reduced levels of Clara cell 10-kDa protein (CC10), a multifunctional secreted protein, naturally produced by the airway epithelia of virtually all mammals. We previously reported that the expression of CC10 is markedly reduced in animals exposed to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, NNK, a potent carcinogen in tobacco smoke. Furthermore, it has been reported that CC10 expression, induced in certain tumor cells, reverses the transformed phenotype. We demonstrate here that NNK exposure of CC10-knock-out (CC10-KO) mice causes a significantly higher incidence of airway epithelial hyperplasia and lung adenomas compared with wild type (WT) littermates (30% CC10-KO versus 5% WT, p = 0.041). We also found that compared with NNK-treated WT mice, CC10-KO mice manifest increased frequency of K-ras mutation, elevated level of Fas ligand (FasL) expression, and increased MAPK/Erk phosphorylation, all of which are considered predisposing events in NNK-induced lung tumorigenesis. We propose that CC10 has a protective role against NNK-induced lung tumorigenesis mediated via down-regulation of the above-mentioned predisposing events.

Investigation of the relationship between cell proliferative activity and invasion, metastasis and prognosis of gastric cancer

Objective: To evaluated Bromodeoxyuridine (BrdUrd)/DNA doubleparametric method in detection of gastric carcinoma and to analyze the relationships of cellular BrdUrd labeling indices(LI), G2/M-phase fraction(G2/MPE) and DNA content to the depth of invasion, lymphatic vessel invasion, lymphatic node metastasis, peritoneal dissemination and blood vessel invasion and prognosis. Methods: Fresh tumor samples from 60 cases were examined by BrdUrd/DNA doubleparametric flowcytometry. Propidium iodide(PI) was used as fluorescent probe for total cellular DNA and a monoclonal antibody against BrdUrd incorporated into DNA. Fluorescein-labeled goat anti-mouse antibody was used as second antibody. Results: The values of BrdUrd LI in patients with serosa invasion was significantly higher than those without serosa invasion (P<0.01); there was statistical significance in 5-year survival rate between the two groups (P<0.01). Both BrdUrd LI and G2/MPF values were significantly higher in patients with lymphatic vessel invasion than those without invasion (P<0.01); the patients with lymphatic vascular invasion carried a significantly poor prognosis (P<0.01). Both BrdUrd LI and G2/MPF values were significantly higher in patients with lymphatic node metastasis than those without metastasis (P<0.01), there was a statistical significance in 5 years survival between these 2 groups. The incidence of lymphatic node metastasis was significantly higher in aneuploid carcinoma (P<0.05), and the patients with aneuploid carried a significantly poor prognosis (P<0.05). Patients with peritoneal dissemination had a significantly worse prognosis (P<0.01). G2/MPF values were significantly higher in patients with blood vessel invasion than those without invasion (P<0.01). Conclusion: Cellular BrdUrd LI, G2/MPF and DNA content are related to depth of invasion, lymphatic vessel invasion, peritoneal dissemination, blood vessel invasion and prognosis of gastric carcinoma.

The prognostic value of proliferation indices: a study with in vivo bromodeoxyuridine and Ki-67.

Proliferation indices are intended to help patients and clinicians make treatment decisions. We have previously demonstrated that a proliferation index based on in vivo labeling of S-phase cells with bromodeoxyuridine (BrdUrd) correlates with Ki-67 labeling index (LI). We now compare the prognostic value of these indices. With written consent, we gave 129 women with biopsy confirmed breast cancer 200 mg/M2 BrdUrd during 30 min immediately preceding surgery. We used IU-4 anti BrdUrd antibody to count the immunohistochemical labeling index (LI) of DNA-incorporated BrdUrd in 2,000 cells and MIB-1 to count Ki-67 (118 cases). Patients received standard surgical and adjuvant treatment. No patients were lost to follow-up and patients were followed a minimum of 2 (median 5.1) years. We compared survival and recurrence in tumors with high vs low labeling indices. We found that women in the low BrdUrd LI group had better disease free survival (92% vs 67% 5-yr DFS p = 0.001) and overall survival (94% vs 70% 5-yr OS, p = 0.0001) than those with a high LI. In comparison, a low Ki-67 index predicted better OS (87% vs 80% 5-yr OS, p = 0.020) and a trend for better DFS (84% vs 72% DFS p = 0.055). The apparent superiority of BrdUrd LI over Ki-67 LI is likely due to chance (p = 0.18). In multivariate survival analyses we found that BrdUrd LI proliferative index significantly improves prediction of DFS or OS even when node status, age or tumor size is in the model. We conclude that markers of proliferation are useful adjuncts in predicting patient prognosis.

Influence of p53 and bcl-2 on proliferative activity and treatment outcome in head and neck cancer patients.

Knowledge about the influence of biomarkers on cell proliferative activity might explain differences in radiosensitivity between head and neck tumors and might improve patient selection for the most optimal treatment strategy. p53 and bcl-2 protein expression were determined immunohistochemically in 56 head and neck cancer patients, treated by surgery only in five cases and by radiotherapy, with or without surgery, in 51 cases. Relationships with various cell proliferation markers, determined by flow-cytometry (G1-phase fraction, S-phase fraction, BrdUrd-labeling index, duration of S-phase and potential doubling time) were investigated. Associations between these cell proliferation parameters, on the one hand, and both p53 and bcl-2, on the other, were not found. Furthermore, p53 and bcl-2 expression were both not related to clinicopathological parameters (T- and N-stage, site, grade) and did not affect loco-regional recurrence-free survival and/or disease-free survival. We could not find a prognostic value for both p53 and bcl-2 protein expression to differentiate radiosensitive from radioresistant head and neck tumors.

Development of gene-switch transgenic mice that inducibly express transforming growth factor beta1 in the epidermis.

Previous attempts to establish transgenic mouse models to study the functions of transforming growth factor beta1 (TGFbeta1) in the skin revealed controversial roles for TGFbeta1 in epidermal growth (inhibition vs. stimulation) and resulted in neonatal lethality in one instance. To establish a viable transgenic model for studying functions of TGFbeta1 in the skin, we have now developed transgenic mice, which allow focal induction of the TGFbeta1 transgene in the epidermis at different expression levels and at different developmental stages. This system, termed "gene-switch," consists of two transgenic lines. The mouse loricrin vector targets the GLVPc transactivator (a fusion molecule of the truncated progesterone receptor and the GAL4 DNA binding domain), and a thymidine kinase promoter drives the TGFbeta1 target gene with GAL4 binding sites upstream of the promoter. These two transgenic lines were mated to generate bigenic mice, and TGFbeta1 transgene expression was controlled by topical application of an antiprogestin. On epidermal-specific induction of the TGFbeta1 transgene, the BrdUrd labeling index in the transgenic epidermis decreased 6-fold compared with controls. Induction of the TGFbeta1 transgene expression also caused epidermal resistance to phorbol 12-myristate 13-acetate-induced hyperplasia, with a reduction in both epidermal thickness and BrdUrd labeling compared with those in controls. In addition, TGFbeta1 transgene expression induced an increase in angiogenesis in the dermis. Given that the TGFbeta1 transgene can affect both the epidermis and dermis, this transgenic model will provide a useful tool for studying roles of TGFbeta1 in wound-healing and skin carcinogenesis in the future.

Morphometric analysis of bromodeoxyuridine distribution and cell density in the rat Dunning prostate tumor R3327-AT1 following treatment with radiation and/or hyperthermia.

To monitor cellular response to single doses of radiation (RT) and/or local tumor hyperthermia (LTH) proliferation kinetics were determined in the anaplastic prostate adenocarcinoma R3327-AT1 grown in Copenhagen rats. Tumor-bearing animals were injected i.v. with a bolus of bromodeoxyuridine (BrdUrd), and at defined times after treatment the tumors were surgically removed, fixed and embedded in paraffin. BrdUrd incorporated into the DNA of S-Phase nuclei was detected on 4-6 microns-thick tissue sections using a monoclonal anti-BrdUrd antibody followed by streptavidin-biotin and alkaline phosphatase as a reporter system. Cell nuclei were stained with the fluorescence dye DAPI (Diaminophenylindole). Morphometric analysis was performed using a computer-assisted Leitz-TAS/plus system. Depending on tumor size, up to 18,000 nuclei were routinely analyzed. Untreated tumors of standardized size (8-10 mm) exhibited a BrdUrd-labeling index (LI) of (6.9 +/- 1.6)%. In general, the LI was higher in the periphery than in the center, being more pronounced in larger tumors. After 6 Gy gamma-rays, the mean LI decreased to 1.8% (24 h) and rose afterwards to 5.4% by 168 h. Following LTH (43.5 degrees C, 35 min water bath), the mean LI rapidly decreased to 2% (8 h), rose to 9.8% (48 h), and plateaued at 6% after 168 h. A combined treatment consisting of irradiation (6 Gy) followed by LTH yielded smallest LI (2.4 +/- 0.18%) and lowest cell density (111 +/- 0.6 nuclei per field) by 168 h. The morphometric procedure was reliable and reproducible and can be used to characterize and compare the effects of different therapies on cell kinetics. Of particular value is that these analyses are done on an intact tissue architecture and hence enable a better interpretation of flow cytometric results of treatment-induced alterations within different topohistological regions in solid tumors. Moreover, the technique provides the basis for 3D reconstruction of the cellular activity and heterogeneity of experimental neoplasms.

The Effects of[formula omitted]-Arginine on Crypt Cell Hyperproliferation in Colorectal Cancer

Background.Colonic crypt cell hyperproliferation characterizes malignant and premalignant conditions of the colon and may be modified by dietary manipulation. This study compared the effect of dietary arginine supplementation on colonic crypt cell proliferation during the initiation and promotion stages of colorectal carcinogenesis.Materials and Methods.One hundred and twenty male Wistar rats were divided into 5 groups of 24 animals each. Groups D, DA, FA, and LA received subcutaneous injections of 1,2-dimethylhydrazine for 20 weeks. Group D received no arginine supplement.l-arginine was given as a 1% solution instead of drinking water to Group DA for 22 weeks, to Group FA for the first 10 weeks, and to Group LA for the last 12 weeks. EDTA animals were given subcutaneous injections of EDTA for 20 weeks. Colonic crypt cell proliferation was assessed in 6 animals from each of the five groups and in 6 normal rats not given DMH or EDTA.Results.The BrdUrd-labeling index and proliferative zone were significantly decreased in all arginine groups (DA, FA, LA). The greatest reduction was evident in Group FA in which tumor incidence and tumor size were also significantly lowered.Conclusions.When given during the initiation phase of carcinogenesisl-arginine significantly reduced colorectal tumor production and crypt cell hyperproliferation.

In vivo bromodeoxyuridine (BrdUrd) labeling index as a prognostic marker in human breast cancer

In vivo bromodeoxyuridine (BrdUrd) labeling index as a prognostic marker in human breast cancer

Cytostatic and apoptotic effects of paclitaxel in human breast tumors.

We have previously reported incomplete cytotoxic responses of other human solid tumors (bladder, head and neck, ovarian and prostate) to paclitaxel. This finding is qualitatively different from the nearly complete response observed in monolayer cultures of human cancer cell lines. The present study examined the pharmacodynamics of paclitaxel in human breast tumors. Three-dimensional histocultures of patient tumors were used. The cytostatic effect was evaluated by measurement of the inhibition of 48-h cumulative bromodeoxyuridine (BrdUrd) incorporation. The apoptotic effect was evaluated in terms of morphological changes and by in situ DNA end labeling. Paclitaxel produced partial cytostasis (approximately 30% maximum) and induced apoptosis (maximum apoptotic index of 3.3% to 29%) in all 15 tumors. More than 95% of apoptotic cells were BrdUrd labeled, but not all BrdUrd-labeled cells were apoptotic. The maximal apoptotic indices in the tumors were significantly correlated with the BrdUrd labeling index of untreated controls (r2 = 0.63, P < 0.01). The maximum apoptotic effect was observed at a tenfold lower drug concentration (0.1 microM) compared to the maximum cytostatic effect (1 microM). Neither of these effects was enhanced by increasing the drug concentration to 10 microM. The pharmacodynamics of paclitaxel in human breast tumors are comparable to those found in other human solid tumors. The labeling of apoptotic cells by BrdUrd and the correlation between the proliferation index and apoptosis suggest that drug-induced apoptosis is linked to cell proliferation and is completed after DNA synthesis. The finding that maximal cytostatic and apoptotic effects of paclitaxel were achieved at or below the clinically achievable concentration of 1 microM suggests further increasing the dose to elevate plasma concentration beyond 1 microM may not improve treatment outcome.

Immediate bromodeoxyuridine labelling of unseparated human bone marrow cells ex vivo is superior to labelling after routine laboratory processing.

It is important to evaluate the proliferation of bone marrow cells in several disease conditions and during treatment of patients with for example cytokines. Labelling with bromodeoxyuridine (BrdUrd), immunocytochemical staining with anti-BrdUrd antibody and analysis by flow cytometry provides a reliable and reproducible technique for estimation of the fraction of cells that incorporated BrdUrd into DNA during S-phase. We have compared immediate BrdUrd labelling of unseparated bone marrow cells with the previously used labelling in the laboratory after routine separation of the mononuclear cells. Bone marrow aspirates from seven lymphoma patients without bone marrow involvement were studied with these two methods. We found higher BrdUrd labelling indices (LI) in the mononuclear cells, when cells were labelled immediately. A large variation in LI was found between patients. Our results suggest that ex vivo BrdUrd labelling of bone marrow cells should be performed immediately after aspiration and before separation, because these data are closer to values reported from in vivo labelling with BrdUrd.

Genetic alterations in lobular breast cancer by comparative genomic hybridization

Infiltrating lobular carcinoma (ILC) and infiltrating ductal carcinoma (IDC) are distinguished by their histopathological appearance. However, little is known about the differences in genetic changes between lobular cancers and ductal cancers. We used comparative genomic hybridization (CGH) and compared aberrations in 19 ILCs and 46 IDCs. The total number of aberrations was lower in ILC than in IDC. While the average number of DNA copy number losses did not reach significance between them, copy number gains were significantly lower in ILCs. Fifteen of 19 ILCs (79%) showed increased copy number of 1q, and 12 cases (63%) revealed loss of 16q. The presence of these aberrations was independent of nodal status, histologic subtypes (pleomorphic or classic ILC), or BrdUrd-labeling index. ILCs had a higher frequency of 16q loss than did ductal cancers, and a lower frequency of 8q and 20q gains. Our data suggest that the altered growth pattern and clinical presentation which characterize infiltrating lobular cancers are correlated with distinct genetic alterations. Int. J. Cancer 74:513–517, 1997. © 1997 Wiley-Liss, Inc.