Paul Galand

Cyclin/PCNA immunostaining as an alternative to tritiated thymidine pulse labelling for marking S phase cells in paraffin sections from animal and human tissues

Abstract Paraffin sections from animal or human tissues fixed in different fixatives were submitted to immunostaining with the mouse monoclonal antibody 19A2, developed by Ogata et al. (1987a) against cyclin/PCNA. Detection of the bound antibody was performed by the indirect method with biotinylated sheep antibody and streptavidin‐biotin‐peroxidase complexes. No, or faint, nuclear staining was seen in material fixed in ethanol, Bouin, Bouin‐Hollande, Carnoy or formaldehyde, whereas readily detectable immunocytochemical reaction was constantly observed over nuclei of methanol‐fixed tissues. Hydrolysis with 2 N HCl prior to immunocytochemistry (as currently performed to render incorporated BrdU accessible to antibodies) somewhat improved the results with Bouin or Carnoy and markedly augmented the intensity of the peroxidase reactions in formaldehyde and in methanol‐fixed tissues.

Cell Renewal in the Human Rectum: In vitro autoradiographic study on active ulcerative colitis

The generation time of the regenerative population in the human rectal mucosa was studied in normal subjects and in patients with ulcerative colitis. The double labeling technique with two pulses of 3H-thymidine, followed by autoradiography, was applied to biopsies incubated in vitro. The results showed that the mean turnover time in ulcerative colitis was significatively shorter than in the normal tissue (34 and 90 hr, respectively; 0.01 > P > 0.001). An extension of the regenerative zone toward the upper part of the crypts was also observed. This suggests that in ulcerative colitis the morphological evolution of the rectal mucosa toward atrophy, which expresses an alteration of the normal equilibrium between cell loss and cell production, is explained by an increased rate of cell desstruction rather than by decreased rate of cell formation.

Comparison of methods based on annexin‐V binding, DNA content or TUNEL for evaluating cell death in HL‐60 and adherent MCF‐7 cells

Abstract. HL‐60 and MCF‐7 cells were treated with 0.15 μM camptothecin (CPT) or with the solvent dimethylsulfoxide (DMSO) for the controls, for 2, 3 and 4 h or for 24, 48 and 72 h, respectively. The apoptotic index (AI) was then evaluated in parallel by the following flow cytometric methods: (1) double staining of unfixed cells with fluoresceinated annexin V and propidium iodide (PI), this after detachment by trypsinization in the case of MCF‐7 cultures; (2) prefixation in 70% ethanol, extraction of degraded, low molecular weight DNA with 0.2 M phosphatecitrate buffer and analysis of the DNA content stained with PI; (3) TUNEL, i.e. labelling of DNA strand breaks with biotin‐dUTP, followed by standing with streptavidin‐fluorescein and counterstaining with PI. In HL‐60 cells, the three methods gave similar results for the AI (3‐4% in the controls and at 2 h of CPT treatment, and 35‐43% at 3 and 4 h after CPT). This indicates that CPT‐induced membrane alteration and DNA fragmentation occurred concomitantly in those cells. For MCF‐7 cells, CPT‐induced apoptosis developed more slowly, the AI, whether based on annexin V or on DNA content, remained unchanged at 24 h, then was increasing to 8% at 48 h and to 25% at 72 h of treatment. In these cells, the TUNEL index did not increase prior to 72 h and the increase was minor (up to 9% vs. 2‐3% in the controls) at 72 h of the treatment. This indicates that in MCF‐7 cells DNA strand breaks cannot be effectively labelled, which may be due to inaccessibility of 3′‐OH ends in the breaks to exogenous terminal deoxynucleotidyl transferase. The mechanism of endonucleolytic DNA fragmentation thus may be different, depending on the cell type.

Cell kinetic indicators of premalignant stages of colorectal cancer

Using an in vitro double labeling technique with two different levels of 3H‐thymidine, the duration of the phase of DNA synthesis (S) and the labeling index (LI) were measured in the colorectal mucosa of three groups of patients: patients with colorectal neoplasms (adenomas and/or adenocarcinomas), patients with inflammatory bowel disease, and a control group of patients without gastrointestinal pathology. In those patients with colorectal neoplasms, samples were obtained from both the neoplastic mucosa and from the normal appearing mucosa at various distances from the lesions. One‐way analyses of variance were used to test the equality of mean S‐phase duration and LI in the various types of tissues. S‐phase duration was significantly longer in the tumor than in the unaffected mucosa of patients with adenocarcinoma (18.65 hours ± 2.3 versus 10.13 hours ± 1.26 P < 0.0001). However, S‐phase duration was significantly longer in the unaffected mucosa of cancer patients than in the mucosa of patients without gastrointestinal pathology (10.58 hours ± 1.84 versus 7.91 hours ± 0.46, P = 0.013). Similarly, LI was significantly higher in the unaffected mucosa of patients with adenoma and adenocarcinoma than in the mucosa of patients without gastrointestinal pathology (19.1% ± 3.0 versus 9.5% ± 2.2, P < 0.0001). There was a highly significant trend to a progressive increase of LI from flat histologically normal appearing mucosas to inflammatory mucosas, adenomas, and adenocarcinomas (P < 0.0001). These results suggest that increased S‐phase duration is specifically related to cancer. In mucosa without histologic sign of malignancy, an increased S‐phase duration would indicate that the malignant process has started. An increased LI would appear to relate to the selective advantage that rapidly proliferating cells hold over less proliferating ones.

Apoptosis in Established and Healing Psoriasis

Background: Previous studies have described apoptosis in the stratum granulosum and in the stratum corneum, but not in the germinative compartment in normal skin. In psoriasis, an increased epidermal apoptosis has been observed in the differentiated compartment, suggesting that apoptosis has a key role in the pathogenesis of psoriasis, as a counteracting factor to the overproduction of cells. Little is known on apoptosis in the germinative compartment. Methods: Apoptosis was studied on biopsies of normal skin, established lesions of psoriasis and PUVA-treated psoriasis using the transferase-mediated uridine nick end labelling method, which detects fragmented DNA, and electron microscopy. Counting of apoptotic cells was restricted to the germinative compartment as defined by Mib1 staining to evaluate the impact of cell loss on cell production and tissue architecture. Results: The apoptotic index was 0.12% in normal epidermis, 0.035% in established psoriasis and 0.31% in regressive psoriasis. Conclusion: These results have three implications: (1) they show the physiological presence of apoptosis in the germinative compartment in normal epidermis; (2) they suggest that induction of apoptosis is involved in the regression of psoriatic hyperplasia after PUVA therapy; (3) the decrease of physiological apoptosis in the psoriatic lesion suggests that this phenomenon could play a role in the induction of psoriatic hyperplasia.

Antisense inhibition of the 27 kDa heat shock protein production affects growth rate and cytoskeletal organization in MCF-7 cells.

MCF‐7 cells were co‐transfected with the human HSP27 antisense cDNA and the neomycin resistance gene, included in the constitutive expression vector pSVL, and the phenotypical changes associated with decreased expression of the HSP27 protein were analysed. Three out of 10 neomycin‐resistant clones obtained proliferated normally and showed a normal HSP27 content (Western blot). The seven other clones (designated as αHSP27 clones) were characterized by a dramatic growth inhibition associated with alterations in cellular morphology. Cells became progressively hypertrophied, exhibited lamellar protrusions and tended to lose contact with each other. They also acquired characteristics of secretory cells, namely the presence of numerous refractile granules and secretory canaliculi. Among the αHSP27 clones, two were immunocytochemically analysed for HSP27 content. Both clones were immunonegative for HSP27, contrary to parental cells and neo transfectants. Actin immunostaining in one of these HSP27 negative clones revealed that microfilament organization changed from diffuse to punctate distribution. Our data support the current concept of a role for HSP27 in cell growth and differentiation and further suggests that this might occur through a control on actin polymerization‐depolymerization.

Cell population kinetics of zymogen and parietal cells in the stomach of mice.

SummaryWith autoradiography after labelling with tritiated thymidine, the kinetics of zymogen and parietal cells were studied in the gastric mucosa of mice. After one intraperitoneal injection of the DNA precursor, zymogen cells in the DNA synthesis phase were clearly identified on autoradiograms, whereas no parietal cells were seen to synthesize DNA.In another group of mice, multiple injections were used in order to obtain a greater number of labelled cells. Following the latter procedure, analysis of grain count distributions over labelled zymogen cells and of labelling indices allowed detection of two subsequent zymogen cell divisions within an interval of approximately two months. This indicates that the cell turnover of zymogen cells is at least partly assured by their own mitotic activity.By contrast, parietal cells showed no evidence of cell division, but appeared to be derived through differentiation from other cells in the neck area of the gland. Analysis of spatial distribution of the labelled parietal cells in the glandular tube indicated that, in time, most newly formed parietal cells undergo a slow migration directed downwards to the bottom of the fundic glands.These results clearly show that the zymogen and the parietal cell population of the fundic glands have a different kinetic behaviour.

Cell cycle parameters in human colon: Comparison between primary and recurrent adenocarcinomas, benign polyps and adjacent unaffected mucosa

The duration of S phase and the labeling index were measured in adenocarcinomas, in polyps and in adjacent unaffected mucosa of a same patient. The in vitro double labeling technique was employed. The S phase duration in tumors was significantly longer than in unaffected mucosa and polyps. This agrees with previously reported data on human skin,16 colon and rectum2 and supports the suggestion that a long S phase might correlated with carcinogenesis. The labeling index in the mucosa adjacent to the tumors is higher than previously measured in normal tissue. Following colectomy however, the labeling index in unaffected mucosa and tumor was lower than prior to surgery. This suggests the disappearance of a systemic factor (in the colon?) which positively regulates the proliferative activity in the rectal and colonic musoca. The maintainance of a lengthened S phase duration in the recurrent tumor where L.I. is lowered, indicates that the proliferation rate and the S phase duration are regulated by separate control factors.

Radioautographic evaluation of the estrogen-dependent proliferative pool in the stem cell compartment of the mouse uterine and vaginal epithelia

Abstract The proliferative pool in the generative compartment of the vaginal and uterine epithelia of spayed mice has been evaluated. Continuous exposure to 3 H-thymidine has been performed by hourly injections of the precursor during 98 hr. This procedure proved to be sufficient to attain a total labelling of the cell populations under study. It is thus clear from our data that homogeneous population of cells continuously proliferate in the basal layer of the vagina, and in the uterine epithelium of the mouse. The proliferation rate of those cells depends on the presence of estrogen, the action of which is to reduce the generative cycle time. In the extreme case of high estrogen doses this accelerating effect of the hormone is accompanied by a marked decrease of the S phase duration. Hence it appears unnecessary to consider, as suggested earlier [2], a stimulative action of estrogen via the awaking of a dormant reserve of cells, whose existence is excluded by our results.

Ki-67 Immunostaining of Normal Human Epidermis: Comparison with 3H-Thymidine Labelling and PCNA Immunostaining

Background: The size of the germinative growth fraction (i.e. the number of actively proliferating germinative cells) of normal human epidermis is still a subject of debate. Ki-67 antigen and PCNA, an auxiliary protein of d-polymerase, are considered as markers of the growth fraction when used under optimal conditions. Method: In the present work, we have compared Ki-67 expression (detected with MIB1 antibody) with PCNA expression (detected with PC10 antibody) in biopsies of normal human epidermis fixed in neutral formalin and using antigen retrieval by microwave processing. To obtain additional information, such as the percentage of cells in S phase, biopsies were also incubated in 3H-thymidine before immunostaining. Results: Before microwave treatment, 8% of the basal cells were positive for MIB1 antibody and 7.8% were positive for PC10 antibody. The 3H-thymidine labelling index was 2.8%. The proportion of MIB1-positive cells rose to 19% after antigen retrieval by microwave processing. In the same way, the 3H labelling index rose to 9%. In contrast, PC10 became positive in all epidermal nuclei. Conclusion: These results suggest that the growth fraction of the germinative cell population of normal human epidermis is not larger than 20% and is composed of cells with a short cell cycle time.