Flow cytometry and in vitro tritiated thymidine labeling in normal rectal mucosa of patients at high risk of colorectal cancer

OBJECTIVES: To compare two different methods to evaluate rectal epithelial cell proliferation as a biomarker of risk of developing colon cancer. METHODS: Samples of normal rectal mucosa from 26 patients at increased risk for colorectal cancer (22 patients with adenoma, three with adenocarcinoma of the large bowel, and one with longstanding ulcerative colitis) were examined by means of in vitro labeling with tritiated thymidine and flow cytometry. RESULTS: We found a significant correlation between thymidine-labeling index and the percentage of cells in S-phase, measured by flow cytometry both in formalin-fixed, paraffin-embedded specimens and in frozen specimens (respectively, r = 0.7647, p < 0.001, and r = 0.4503, p < 0.01). However, using flow cytometry, the percentage of cells in S-phase was significantly higher than the thymidine-labeling index in both fixed-embedded and frozen specimens (p < 0.01). Proliferative parameters were not higher in patients with colon carcinoma, and were not related to the degree of dysplasia, the number of adenomas, or familial occurrence of colorectal cancer. Two specimens taken from normal rectal mucosa of two patients with adenomas showed aneuploidy. No aneuploidy was found in normal rectal specimens of patients with adenocarcinoma. CONCLUSIONS: These results show that the calculation of cells in S-phase with in vitro tritiated thymidine labeling or by flow cytometry produces different results. However, the significant correlation between corresponding parameters obtained with these techniques support the use of either method as "intermediate biomarkers" of colorectal cancer risk and prognosis.     

Effort and achievement in national family planning programmes

Cell proliferation of 174 specimens obtained from the primary gastric cancers using endoscopic biopsy was investigated by immunohistochemical analysis with the monoclonal antibody PC10, which recognizes a proliferating cell nuclear antigen (PCNA) in formalin-fixed and paraffin-embedded material. All the examined samples showed nuclear staining for PCNA in cancer cells. The investigation was to test the correlation between PCNA labeling and lymph node metastasis. DNA aneuploidy was often encountered in tumors with nodal involvement and lymphatic invasion. The logistic regression analysis identified PCNA labeling rates (LRs), tumor size, and macroscopic type as independent significant factors for lymph node metastasis. When the PCNA LRs and clinicopathologic parameters were entered into the Cox regression analysis, PCNA LRs and DNA ploidy emerged as independent significant prognostic factors. In addition, combination assay of PCNA LRs and DNA ploidy yielded a powerful prognostic indication for patients with gastric cancer.     

Oral calcium inhibits rectal epithelial proliferation in familial adenomatous polyposis

Calcium reduces colorectal cell turnover and might therefore protect against neoplasia. The inhibitory effects of dietary calcium were tested in a double-blind controlled trial in patients with familial adenomatous polyposis who had undergone previous abdominal colectomy and ileorectal anastomosis. Patients received supplemental calcium carbonate (1500 mg/day) or placebo tablets for 6 months; sigmoidoscopy was performed before and after treatment. Rectal biopsies were maintained in short-term organ culture, and crypt cell production rate (CCPR) was measured stathmokinetically. A total of 25 patients completed the trial; polyp counts were obtained before and after treatment in all and CCPR values in 16. Calcium treatment reduced the mean (s.e.m.) CCPR from 4.72 (0.48) to 2.42 (0.48) cells per crypt per h (P < 0.05), while values for placebo were unchanged (5.46 (1.21) versus 5.08 (1.17) cells per crypt per h). Calcium had no demonstrable effect on the number, size or distribution of rectal polyps. The ability of oral calcium supplementation to suppress rectal epithelial proliferation supports its potential to prevent development of colorectal carcinoma in high-risk individuals.     

Application of biotin, digoxigenin or fluorescein conjugated deoxynucleotides to label DNA strand breaks for analysis of cell proliferation and apoptosis using flow cytometry

A flow cytometric method has recently been developed using biotinylated dUTP (b-dUTP) in a reaction catalyzed by terminal deoxynucleotidyl transferase (TdT) to identify the endonuclease-induced DNA strand breaks occurring during apoptosis. Counterstaining of DNA makes it possible to relate apoptosis to cell cycle position or DNA index. In the present study, we compared this method with one using digoxigenin-conjugated dUTP (d-dUTP) to label apoptotic cells. The discrimination of apoptotic from nonapoptotic cells was similar when incorporation of d-dUTP was compared with b-dUTP. Both techniques resulted in a 20-30 fold increase in staining of apoptotic over nonapoptotic cells although somewhat less background fluorescence was observed with the d-dUTP. Direct labeling with fluoresceinated dUTP (f-dUTP) was less sensitive in detecting DNA strand breaks, but had the advantage of simplicity. The principle of labeling DNA strand breaks using TdT was also employed to identify DNA replicating cells. To this end, the cells were incubated in the presence of BrdUrd, then exposed to UV light to selectively photolyse DNA containing the incorporated BrdUrd. DNA strand breaks resulting from the photolysis were then labeled with b-dUTP or d-dUTP. This approach is an alternative to immunocytochemical detection of BrdUrd incorporation, but unlike the latter does not require prior DNA denaturation, thus can be applied when the denaturation step must be avoided. The method was sensitive enough to recognize DNA synthesizing cells that were incubated with BrdUrd for only 5 min, the equivalent of replication of less than 1% of the cell's genome. The discrimination between apoptotic vs. BrdUrd incorporating-cells is based on different extractability of DNA following cell fixation. This method can be applied to analyze both cell proliferation (DNA replication) and death (by apoptosis) in a single measurement.     

Measurement of cell proliferation by labeling of DNA with stable isotope-labeled glucose: studies in vitro, in animals, and in humans

A method for measuring DNA synthesis and, thus, cell proliferation, in vivo is presented. The technique consists of administering [6,6-2H2]Glc or [U-13C]Glc, isolating genomic DNA, hydrolyzing enzymatically to free deoxyribonucleosides, and derivatizing for GC-MS analysis of dA or dG isotopic enrichments, or both. Comparison of dA or dG to extracellular Glc enrichment (with a correction for intracellular dilution) reveals the fraction of newly synthesized DNA, by application of the precursor-product relationship. Thus, the technique differs from the widely used [3H]thymidine or BrdUrd techniques in that the de novo nucleotide synthesis pathway, rather than the nucleoside salvage pathway, is used to label DNA; the deoxyribose rather than the base moiety is labeled; purine rather than pyrimidine deoxyribonucleosides are analyzed; and stable isotopes rather than radioisotopes are used. The method is applied here in vitro to the growth of HepG2 and H9 cells in culture; in animals to proliferation of intestinal epithelium, thymus, and liver; and in humans to granulocyte turnover in blood. In all instances, measured cell proliferation kinetics were consistent with expected or independently measured kinetics. The method has several advantages over previously available techniques for measuring cell turnover, involves no radioactivity or potentially toxic metabolites, and is suitable for use in humans. The availability of a reliable and safe method for measuring cell proliferation in humans opens up a number of fundamental questions to direct experimental testing, including basic problems related to cancer, AIDS, and other pathologic states.     

Long-term depression of excitatory synapses in the cortex and hippocampus

During a 3-year period, 258 infants and children underwent rectal biopsy to exclude Hirschsprung's disease (HD) and related disorders; 32 (12%) were found to have HD. Major morbidity occurred in 3 (2%) of 148 patients undergoing rectal suction biopsy (RSB) and 22 (13%) of 168 suction biopsies were inadequate for diagnosis. In 102 children over 6 months of age, Storz rectal cup biopsy forceps were used with no significant morbidity and adequate biopsies were obtained in 96% of cases. Open rectal biopsy was performed in 8 patients. The RSB tube is safe and reliable, but attention to technique is important. For children over 6 months of age undergoing rectal biopsy for HD, the Storz rectal cup biopsy forceps yields superior results.     

Disseminated infection due to Chrysosporium zonatum in a patient with chronic granulomatous disease and review of non-Aspergillus fungal infections in patients with this disease

BACKGROUND: Insulin-like growth factor-2 (IGF-2) is considered one of the autocrine growth factors in colorectal carcinoma. In addition, it is well known that IGF-2 is produced in the liver. However, the role of IGF-2 in liver metastasis is not yet understood clearly. METHODS: Immunohistochemical staining of IGF-2 and IGF-1 receptor (IGF-1R) was performed on tissue samples of liver metastases from 30 colorectal carcinoma patients. In situ hybridization of IGF-2 also was conducted on the same tissue samples. Furthermore, proliferating cell nuclear antigen (PCNA) was immunohistochemically stained for use as an indicator of the proliferative activity of cancer cells. RESULTS: Invasive margins of liver metastases were stained highly by both IGF-2 (70%) and IGF-1R (83%). Overexpression of IGF-2 protein and mRNA was observed in the normal liver adjacent to the tumor. The PCNA labeling indices (LIs) of the IGF-2 positive groups were significantly higher than those of the IGF-2 negative group (P < 0.0001). In addition, the PCNA LIs for the IGF-1R positive groups also were significantly higher than those for the IGF-1R negative group (P=0.0002). CONCLUSIONS: These findings suggest that hepatocyte-derived IGF-2 stimulates tumor cell proliferation by a paracrine mechanism and plays an important role in tumor progression in colorectal carcinoma patients with liver metastases.     

Accelerated gastric epithelial proliferation

Gastric body mucosal proliferation was quantified and localised under conditions of increased gastrin drive using a variety of techniques. Rats were given omeprazole 400 mumol/kg/day by gavage and after 30 days mean serum gastrin rose 11-fold (p < 0.001). Total mucosal polyamines rose 220% from 15.9 to 50.9 nmol/mg protein (p < 0.001). This was associated with a 238% increase in crypt cell production rate from 0.541 to 1.83 crypt cells/h by vincristine metaphase arrest (p < 0.02). Using computer aided counting of proliferating cell nuclear antigen (PCNA) immunostained nuclei to assess epithelial proliferation in hypergastrinaemia rat stomach: mucus neck cell PCNA labelling was increased by 41% (p < 0.001) and gland cell PCNA labelling was increased by 222% (p < 0.001). PCNA/AgNOR (argyrophilic nuclear organiser regions) co-stained sections were used to assess proliferative activity in cycling and non-cycling cell populations. Data from these experiments suggest that, in addition to increasing the number of mucosal cells in cycle, cell life and cell cycle duration may be reduced in hypergastrinaemia.     

Computerized determination of proliferating cell nuclear antigen expression in meningiomas. A comparison with non-automated method

Proliferating cell nuclear antigen (PCNA) expression has been proven to be a significant marker of cell proliferation in meningiomas, which correlates with growth rate and, as shown by several authors, possibly provides prognostic information concerning biologic behavior. However, the current method for determining PCNA labeling index (LI) is tedious and time consuming like all the nonautomated methods for evaluating cell kinetics, presenting high interobserver and interlaboratory variability and low reproducibility. In the present study, we introduce a semi-automated computer-assisted image analysis method for determining PCNA LI in 38 meningiomas, in parallel with the current nonautomated method. Image analysis technique permits unbiased cell counting, standardizes the degree of staining intensity and provides instant results. By calculating coefficient of variability, the method proved to be highly reproducible. The correlation between the results provided by the nonautomated and the semiautomated image analysis method showed a high agreement between them, with a correlation coefficient, r, of 0.82. In conclusion, we consider that image analysis contributes to the accuracy, reproducibility, and practicality of PCNA LI determination so that along with other useful parameters this significant marker may serve to predict the clinical behavior in meningiomas.     

Long-term prognostic value of PCNA labeling index in primary operable breast cancer

Cell proliferation was evaluated in 167 tissue specimens obtained from primary breast cancer patients who had undergone radical surgery between 1984 and 1988. Formalin-fixed and paraffin-embedded tissue specimens were used in the immunohistochemical study. The immunohistochemical method was carried out using the avidin-biotin immunoperoxidase technique, and anti-proliferating cell nuclear antigen (PCNA) monoclonal antibody was used for primary antibody. Based upon the PCNA labeling index (LI), the patients were divided into two groups: low PCNA, <25% and high PCNA, >/= 25%. The PCNA LI ranged from 1% to 76% (mean, 23.9%). Patients aged 50 years. There was no relationship between the PCNA LI and tumor size, lymph node involvement and hormone receptors. The survival curves of 146 invasive breast cancer patients showed that the high PCNA group had poor overall survival compared with the low PCNA group. A significant difference in the overall survival between the high and low PCNA groups was observed in lymph node-positive patients, however, no significant difference was found between the two groups in lymph node-negative patients. PCNA LI was identified as an independent predictor in primary breast cancer patients.     

Regulation of PTP-1 and insulin receptor kinase by fractions from cinnamon: implications for cinnamon regulation of insulin signalling

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.     

Increased apoptosis accompanies neoplastic development in the human colorectum

A disturbance in the balance between cell proliferation and cell loss, or apoptosis, may underlie neoplastic development. Therefore, we determined spontaneous apoptotic and proliferative rates in normal, hyperplastic, adenomatous, and malignant colorectal epithelia. In paired sections, DNA strand breaks were detected using the terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling assay, and apoptotic cells were also identified in H&E-stained slides by morphological criteria. Cell proliferation, bcl-2, and p53 expression were analyzed using specific monoclonal antibodies. In normal mucosa, luminal epithelial cells demonstrated higher rates of apoptosis compared to cells in the proliferative zone. Neoplastic transformation was associated with a significant increase in rates of apoptosis and proliferation. However, apoptosis, but not proliferation, decreased at the adenoma-to-carcinoma transition coincident with expression of mutant p53. In carcinomas, both mutant p53 and bcl-2 protein levels were associated with attenuated apoptotic rates. In conclusion, apoptosis is an important regulator of growth in normal and neoplastic colorectal epithelia. Increased apoptosis and proliferation accompany neoplastic transformation, suggesting that an alteration in apoptotic rates is an important event in colorectal carcinogenesis. Furthermore, the imbalance in these processes found in carcinomas may facilitate tumor growth and progression.     

Phototherapy of cancer and atheromatous plaque with texaphyrins

The incidence of nonmelanoma skin cancer, including both squamous cell carcinoma and basal cell carcinoma, is a significant health problem in the United States. Actinic keratosis (AK), the precursor of cutaneous squamous cell carcinoma, is a major risk factor for nonmelanoma skin cancer. In addition, AKs are tissue targets for the identification of biomarkers for use in chemopreventive studies. The biomarker addressed in this study is epidermal cell proliferation, as quantitated by proliferating cell nuclear antigen (PCNA). Shave biopsies were obtained from AKs, tissue immediately adjacent to AKs, normal-appearing, upper-medial arm skin, and non-sun-exposed skin from 19 subjects. When any degree of PCNA staining was considered positive (semiquantitative 1-4 scale), there was a significant difference and a progressively increasing mean PCNA labeling index (LI) in the total epidermis (basal and suprabasal layers), beginning with non-sun-exposed buttock skin, with the lowest LI (2.5 + or - 1.6%), followed by upper-medial arm skin (12.3 + or - 7.4%; P = 0.0015), skin adjacent to AKs (19.2 + or - 12.2%; P = 0.0218), and finally, AKs with the highest LI (34.6 + or - 20.1%; P = 0.0017). This same pattern was observed when the epidermis was separated into basal and suprabasal layers, with the exception of a nonsignificant result for upper-medial arm skin compared with adjacent skin in the basal layer (P = 0.3981). PCNA LIs were also analyzed separately by staining intensity (i.e., scores of 1-4). The PCNA LI in skin with varying degrees of sun damage and/or histological atypia is a candidate surrogate end point biomarker for skin cancer chemoprevention studies.     

An observation of the effect of tanshinone on cancer cell proliferation by Brdu and PCNA labeling

The aim of this study was to find out the anti-cancer activity of Tanshinone and its mechanism of action. Human hepatic carcinoma cell line (SMMC-7721) and leukemia cell line (HL60) were treated with tanshinone the cancer cell proliferation indices were measured by Brdu Labeling and immuno-histochemical stain of PCNA. The Brdu labeling rates of human hepatic carcinoma and leukemia cells treated with tanshinone were 8.95% and 19.01%, which were lower than those of controls (28.0%, 25.57%) respectively (P < 0.01), PCNA positive rates were 57.0% and 30.32%, which were significantly lower than those of controls (74.3%, 47.05%) (P < 0.01). The results indicate that Brdu labeling and PCNA detection may have important utility in the studies of tumor cell proliferation and the relative factors affecting the cell proliferation. The inhibitory effect of tanshinone on cancer cell proliferation might be associated with inhibiting DNA synthesis, PCNA expression and activity of DNA polymerase delta of the tumor cells.     

Colorectal cell kinetics

The assessment of cell proliferation in colorectal tissue may provide information with both prognostic and therapeutic implications. A variety of methods are available, including flow cytometric estimations of S phase fraction, immunohistochemical and autoradiographic visualization of exogenous and endogenous proliferation proteins, and morphological and stathmokinetic techniques. There is some correlation between Dukes stage and proliferation state features, and there is increased proliferative activity throughout the adenoma-carcinoma sequence. Data on cell proliferation rates are difficult to obtain. When correctly applied, the metaphase arrest technique remains the 'gold standard' of measuring proliferation, but its usefulness in clinical practice is limited. Recent studies have employed dual measurement flow cytometry and double labelling techniques to produce rate data.     

Analysis of proliferative activity using anti-proliferating cell nuclear antigen antibody in gastric cancer tissue specimens obtained by endoscopic biopsy

BACKGROUND: Proliferative activities of tumors are thought to be prognostic features of malignant tumors, but their value as measured by proliferating cell nuclear antigen (PCNA) labeling remains unclear in gastric cancer. METHODS: PCNA labeling rates (LR) were quantified in 121 paraffin-embedded biopsy specimens from primary tumors by immunocytochemistry. RESULTS: Immunohistochemical analyses have demonstrated that PCNA presents an intense staining in the nuclei of tumor cells and mucous neck cells of gastric glands. The PCNA LR ranged from 12% to 79% (mean +/- standard deviation), and a significant correlation was found between bromodeoxyuridine labeling indices and PCNA LR: PCNA LR were closely associated with tumor size, serosal invasion, and nodal involvement. The patients with tumors with high PCNA LR (greater than 40%) were dead significantly earlier than were those with tumors with low PCNA LR: When the PCNA LR and all the clinicopathologic parameters were entered into a Cox regression model, PCNA LR emerged as an independent prognostic factor. CONCLUSION: These results indicate that PCNA LR may be a potentially useful prognostic factor for gastric cancer.     

Accumulation of 5-aminolevulinic acid-induced protoporphyrin IX in normal and neoplastic human endometrial epithelial cells

The aim of this study was to evaluate 5-Aminolevulinic acid (ALA)-induced fluorescence of normal and neoplastic endometrial epithelial cells for diagnosis and photodynamic treatment. Fluorescence of ALA-induced PpIX in vitro was measured by flow cytometry in two different human endometrial adenocarcinoma cell lines and in normal cells cultivated from fresh endometrial tissue of three premenopausal patients. The cells were analysed after incubation with different concentrations of ALA during 3, 6, or 24 hours. Both tumor cell lines showed a statistically significant higher fluorescence of PpIX than normal epithelial cells after incubation with 1 mg ALA per ml medium during 24 hours. The well-differentiated cancer cells produced significantly more PpIX than the poorly differentiated cancer cells. Relative PpIX intensity of the two cancer cell lines correlated with cell proliferation rate as measured by the doubling times of the cells. Higher accumulation of Pp IX in neoplastic endometrium compared to normal endometrial epithelial cells may provide targeted biopsies and selective photodynamic destruction of neoplastic micro-lesions.     

Regional differences in bromodeoxyuridine uptake, expression of Ki-67 protein, and nucleolar organizer region counts in glioblastoma multiforme

To investigate intratumoral differences in indices of tumor cell proliferation, we measured the bromodeoxyuridine labeling index (BrdUrd LI), the Ki-67 protein proliferating cell indices (PCIs) determined by monoclonal antibody MIB 1 in microwave-processed paraffin sections (MIB 1 PCI) and in some cases by monoclonal antibody in frozen sections (Ki-67 PCI), and counts of argyrophilic nucleolar organizer regions (AgNORs) in 20 glioblastomas. In the most actively proliferating areas, MIB 1 and Ki-67 PCIs correlated well with the BrdUrd LI and with each other, while AgNOR counts correlated less strongly with these indices. In less active areas, the MIB 1 PCI and BrdUrd LI changed concomitantly from one area to another within a tumor except in areas of pseudopalisading with necrosis; in these areas the BrdUrd LI decreased significantly compared with neighboring tumor tissue, while the MIB 1 PCI did not. There was very little staining of gemistocytic nuclei with either anti-BrdUrd or MIB 1 monoclonal antibodies; this supports the concept that gemistocytes are mainly quiescent cells. AgNORs in all of the above-mentioned areas varied from tumor to tumor, which suggests that they may indicate some cellular activity other than proliferation. The close correlation between the BrdUrd LI and Ki-67 protein PCIs in corresponding regions of glioblastomas suggests that MIB 1 staining of microwave-processed paraffin sections can be used to evaluate the growth potential of individual glioblastomas and possibly of other gliomas as well.     

Expression of proliferating cell nuclear antigen(PCNA) in biopsy and autopsy specimens of gastric carcinoma

Although proliferating cell nuclear antigen (PCNA) is known to be an indicator of malignant potential in tumors, the biological and clinicopathological significance of PCNA in tumor tissue is controversial. METHODS: Immunohistochemical expression of PCNA was examined in 58 gastric carcinoma tissues obtained at autopsy to test the clinicopathological significance. In addition, in 24 of the 58 tumor tissues we compared immunohistochemical expression of PCNA in biopsy and autopsy specimens from the same patient in order to know whether the proliferating activity of tumor cells is stationary from the early stage to the end of tumor growth. RESULTS: 1. PCNA was undetectable in some tumor tissues (12.5% in biopsy and 10.3% in autopsy specimens). 2. the frequency of PCNA positive cases and labeling index (LI) (%) of PCNA in tumor tissues were not significantly different between biopsy and autopsy specimens. 3. the intensity of PCNA reaction was not related to prognosis. 4. PCNA positive cases and LI did not correlate with survival condition. CONCLUSION: It is hard to say whether PCNA is a reliable indicator in predicting malignancy and prognosis of gastric cancer.