Amado F. Badrán

CIRCADIAN RHYTHM OF DNA SYNTHESIS AND MITOTIC ACTIVITY IN TONGUE KERATINOCYTES

Tongue keratinocytes have a high mitotic index (MI) with an evident circadian variation. Our study set out to compare and contrast two phases of the cell cycle: DNA synthesis (S‐phase), with inmunocytochemical detection by bromodeoxyuridine (BrdU), and mitosis (M‐phase), by the colchicine‐arrest of metaphase method, exploring both the dorsal and ventral surfaces of the mouse tongue throughout a circadian period. Adult male mice standardized for light periodicity used for MI experiment were injected intraperitoneally with colchicine. Other animals were injected intraperitoneally with 5‐BrdU for S‐phase determination. Animals given both treatments were divided into six groups and killed at 4h intervals until 20:00h. Tongue samples were processed for histology and immuno‐histochemistry. S and M indices were expressed as labelled nuclei or colchicine metaphases, respectively, per 1000 nuclei. Peak MI occurred at 12:00, with the minimum value at 20:00 on dorsal and ventral tongue surfaces. Peak S‐phase was at 04:00, whereas the minimum value was at 16:00 for both surfaces. These results show that the proliferative activity of the tongue epithelium is of similar intensity and temporal distribution on both surfaces.

VARIATIONS IN DNA SYNTHESIS AND MITOTIC INDICES IN HEPATOCYTES AND SINUSOID LITORAL CELLS OF ADULT INTACT MALE MOUSE ALONG A CIRCADIAN TIME SPAN

Variations of DNA synthesis (DNAS) and mitotic indices along a circadian time span are described in the hepatocyte and sinusoid litoral cell populations of adult intact male mouse liver. Standardized (light from 0600 to 1800) mice were killed in groups of six to nine animals, every 2-4 hr along a circadian time span. Hepatocytes show significant peaks in the synthesis of DNA and the mitotic activity at 0200 and 1400, respectively. These results correspond to those previously described by us in young immature liver, regenerating liver and hepatomas. The phase differences between these peaks and the differences between their absolute values are discussed. Also considered are the practical consequences of our findings for experimental design. The curve of DNA synthesis of sinusoid litoral cells show a peak at 0200. The mitotic index show a bimodal waveform with peaks at 0800 and 2000. The existence of four different cell populations composing the so called sinusoid litoral cells and also the migration into and out of the liver of some macrophages considered as litoral (Kupffer) cells in our counts, makes interpretation of the curves somewhat complicated and deserves further analysis.

MITOTIC ACTIVITY OF THE DUODENAL CRYPT ENTEROCYTES IN MICE TRANSPLANTED WITH EA21A MAMMARY CARCINOMA

The presence of a tumor generally changes the mitotic activity of the normal cell population in mice. In the present work, the mitotic activity of the duodenal crypt enterocytes in EA21a mammary carcinoma‐bearing mice was determined. The results show that there is a patent circadian variation in normal mice and, in the presence of the EA21a mammary tumor, cell proliferation is stimulated. Stimulation was evident in enterocytes from the intermediate as well as the superficial regions of the crypt. Some humoral factors produced by the transplanted tumor could interfere with the regulatory mechanism of the mitotic activity of duodenal crypt enterocytes.

Sex- and age-related temporal variations in intestinal-epithelium proliferation in the suckling mouse.

Intestinal-crypt enterocytes are a cell population undergoing constant renewal in the mouse. Both adult and 28 d old animals have been shown to exhibit circadian rhythms in cell proliferative indices, but there are only scant data on the 24 h mitotic activity in the small and large intestine of younger mice. The present studies were thus undertaken in order to characterize the proliferative pattern of enterocytes in the duodenum and colon of 7 and 14 d old males and females of the C3H/S strain. Animals of each sex and from each age group were sacrificed every 4 h during a 24 h span, with each animal receiving an injection of colchicine 4 h before sacrifice. Samples of duodenum and colon were removed and processed for hematoxylin–eosin staining. Twenty longitudinally sectioned crypts within each sample were analyzed, and the mitotic indices of both cell populations from each animal were estimated. The arithmetic mean±SEM for each experimental group were then calculated and the statistical significance of differences between the means assessed by ANOVA and Student t-tests. We observed a greater daily mitotic activity in the duodenum than the colon, and moreover enterocytic proliferation in both those regions was greater in 14 than 7 d old animals. Twenty-four hour variations in mitotic activity occurred in all the experimental groups and tissues except for the large intestine of 7 d old females. Finally, the temporal profile of epithelium proliferation in the suckling mouse varied with age, sex, and site of the intestine studied.

Cytokeratin immunoreactivity in mouse tissues: study of different antibodies with a new detection system.

The cross-reactivity of a group of monoclonal antibodies (MABs) generated against human cytokeratins (CKs) was investigated in mouse tissues. Formalin-fixed and paraffin-embedded sections of lung, stomach, small and large intestine, liver, and kidney were immunostained with MABs after epitope retrieval with enzyme digestion. AE1/AE3, a “cocktail” of two MABs that recognizes basic and acidic CKs, 5D3 MAB to low molecular weight CKs (8, 18, and 19), and monospecific MABs to CK 7 and 20 were tested. Additionally, CK 17 and 34&bgr;E12 MABs to high molecular weight CKs were evaluated in the same organs and in sections from skin and preputial glands. We employed the new universal animal system (ARK) as the detection system. The results showed intense reactivity for the first group of antibodies used, with topographic distribution similar to that in human tissues, with the exception of CK 7 in lung parenchyma, which displayed reactivity only in type II pneumocytes, with negativity of adjacent bronchial epithelium. Also of note was the lack of reaction of liver hepatocytes and renal tubular cells to AE1/AE3 and 5D3 MABs. Regarding the second group of antibodies, no reaction was obtained for CK 17 in the tissues tested. On the contrary, 34&bgr;E12 MAB yielded intense reactivity in cells of epidermis and hair follicles. Compared to other detection systems used previously in this animal, ARK produced a well-defined reactivity at the cellular level without any background. We conclude that a useful panel of anti-CK antibodies commonly used in human pathology can be applied successfully to mouse tissues after enzyme digestion, leading to a more accurate definition of cellular populations in this laboratory animal.

MITOTIC ACTIVITY OF THE PARS INTERMEDIA IN THE FEMALE MOUSE: AGE-ASSOCIATED VARIATIONS IN PROLIFERATION RATE AND CIRCADIAN PERIODICITY

We previously reported daily variations in the mitotic activity of the endocrine cells in the pars intermedia of 21- and 28-day-old male mice. Since cellular proliferation might be affected by factors such as sex and age, we undertook the present experiments to study the mitotic activity of the pars intermedia from 14-, 28-, and 150-day-old female mice. Inbred C3H/S mice, grouped according to age, were housed under standard conditions (12h each of light and dark [LD 12:12]) for periodicity analysis and were killed in lots of 5–11 animals every 4h over a single 24h cycle, with each mouse receiving 2 μg/g of colchicine 4h before decapitation. Pituitaries were excised, extracted, fixed in buffered formaldehyde, embedded in celloidin-paraffin, sectioned at 5 μm, and stained with hematoxylin and eosin. We counted the total number of nuclei to estimate the total number of cells monitored and then calculated the mitotic index (metaphases/1000 nuclei). Differences were analyzed for statistical significance by the Student t test. While the 14-day-old animals manifested no significant changes in mitotic activity during the 24h cycle, the 28- and 150-day-old mice showed higher mitotic indices during the period of darkness. The average mitotic activity over the entire cycle, however, was higher in the two groups of younger animals than in the 150-day-old mice. Moreover, the averages for the 28-day-old females were higher than the corresponding values previously reported by us for male mice of the same age. (Chronobiology International, 17(6), 751–756, 2000)

Daily variations in colchicine-induced apoptosis in duodenal crypts.

Apoptotic cell death can be induced by several agents, among them colchicine, a microtubule disrupting‐drug that affects continuously renewing cell populations, such as the intestinal crypt enterocytes. The objectives of this investigation were (1) to confirm in vivo colchicines‐inductive effect and (2) to determine the existence of 24 h variations in the crypt enterocytes apoptotic indices. The study was done on C3H/S male adult mice housed under standardized conditions. Starting at midnight until the end of a circadian period, subgroups of mice were sacrificed after having been injected with colchicine or saline i.p. 4 h beforehand. Duodenal samples were processed for hematoxylin‐eosin staining and TUNEL technique. In order to score the number of apoptosis, the longitudinal sections of the crypts were divided into three regions comprised, respectively, of tiers 1–4, 5–12, and 13–20, proceeding from the bottom to the top of the crypt. Values of each lot were expressed as mean±SEM. A highly significant statistical difference in apoptotic indices was found for colchicine‐treated animals. The 24 h curve for colchicine‐induced apoptosis displayed qualitative and quantitative differences compared to other inducer agents. Highest apoptotic indices were found in the deepest crypt regions. Daily variations were observed in all the crypt sectors of the colchicine‐treated animals and in tiers 5–12 of the saline controls. The present work demonstrates that the colchicine cytotoxicity due to its apoptotic‐inducing effect depends on the dosing time during the 24 h in this mouse strain.

Ki67 reactivity in cell membrane and cytoplasm of tonsil crypt epithelial cells.

To the Editors: The unexpected finding of selective but nonspecific reactivities of monoclonal antibodies has been the subject of several articles (1,2). Changes in epitope structure caused by fixation and the existence of short determinants shared by unrelated peptides and proteins have been pointed out, among others, as the possible sources of the anomalous reactions observed (1,3). In a recent article, Hirokawa and Carney (4) reported on the cell membrane and cytoplasmic reactivity for MIB-1, a wellknown Ki67 monoclonal antibody, in hyalinizing trabecular adenoma of the thyroid gland, emphasizing its value as a diagnostic tool in the differential diagnosis with papillary carcinoma. They also observed a faint reactivity in the apical surface of some normal follicular cells. Other normal tissues were not included in the study. Recently, we observed a similar immunoreactivity in squamous epithelial cells of the tonsil crypts, working with a different anti-Ki67 monoclonal antibody (Ki88; Biogenex, San Ramon, CA). With the introduction of this new clone in our laboratory, we performed a test battery to determine the optimal antibody dilution, and the best methods of antigen retrieval and immunodetection. To that end, we selected formalin-fixed, paraffinembedded tonsils currently used as controls for immunostaining of hematolymphoid (CD20, CD3, CD43, etc.) and proliferative markers (KiS5, MIB5). Several 5mthick sections were placed in a plastic container with citrate buffer (pH 6) and warmed in a water bath at 95°C to 99°C for 30 minutes. Ki88 monoclonal antibody (1/10–1/20) was incubated at room temperature for 1 hour. Two avidin-biotin-peroxidase methods (Super Sensitive, Biogenex, and Vectastain, Vector, Burlingame, CA) were used as detection systems, with DAB as chromogen. The results showed nuclear/nucleolar immunopositivity in germinal center cells of lymphoid follicles, as well as in basal epithelial cells of the superficial squamous lining (Fig. 1). Unexpectedly, many epithelial cells deep in the crypts forming a lymphocyte-containing mesh exhibited strong and delicate reactivity in the cell membranes and also in their cytoplasms (Fig. 2) No comparable staining was observed in the squamous superficial lining (Fig. 2) nor in other tissue components. Our findings support the view of Hirokawa and Carney that there is a nonrelated antigen restricted to some epithelial cells, probably containing epitopes of similar sequence (3,5), that crossreacts with Ki67 antibodies represented by MIB-1 in their article, and by Ki88 in the current study. This observation reminds us the CD79a crossreactivity in megakaryocytes produced by HM57 antibody, which has been recently evaluated as a marker for this cellular type in bone marrow biopsies (6). Other tissues should be tested for Ki67 immunopositivity to determine the real spectrum of this anomalous reactivity and its biologic significance.