Effects of aluminum on rat bone cell populations.

Aluminum (Al) loading is associated with reduced bone formation and osteomalacia in human and certain animal models. However, uncertainty exists as to the cellular effect(s) of Al as both inhibition and stimulation of osteoblast proliferation have been reported. Furthermore, the extent to which Al affects osteoprogenitor cell populations is unknown. To determine the cellular effects of Al in the rat, an animal model in which Al bone disease has been produced, we compared the in vitro effect of 10-50 microns Al on the proliferation and hydroxyproline collagen formation of marrow osteoprogenitor stromal cell populations and perinatal rat calvarial osteoblasts. In subconfluent cultures, Al suppressed proliferation of both marrow fibroblast-like stromal cells and calvarial osteoblasts. In confluent cultures, however, Al selectively stimulated periosteal fibroblast and osteoblast DNA synthesis and collagen (hydroxyproline) production, both in the presence or absence of 1,25-dihydroxyvitamin D. Osteocalcin was not detected in osteoblast-conditioned media or extracellular matrix. These observations suggest that the bone formation defect associated with Al toxicity in growing rats may be a function of impaired patterns of osteoprogenitor/osteoblast proliferation. Furthermore, the Al-stimulated increase in collagen formation is consistent with the development of osteomalacia in Al-toxic humans and animals. The mechanism by which Al stimulated DNA synthesis and collagen production in more mature cultures awaits further study.

Persistence of the circadian variation and altered response to hepatectomy of hepatic ornithine decarboxylase activity with malignant tumor burden.

We measured the effect of MC-26 mouse colon cancers (of different sizes) on the circadian rhythm of hepatic ornithine decarboxylase (ODC) activity and hepatic ODC activity during the 24 hr after 60% hepatectomy. Tumor-free control mice showed a normal circadian rhythm of ODC activity with the highest levels at 1100 hr and the lowest levels at 2300 hr. The amplitude of the rhythm was diminished significantly in mice with a large tumor burden (3% of their body weight), and hepatic ODC activity was significantly less than in the tumor-free mice at every point during the 24 hr of the study. In mice with "early" tumors (0.3% of body weight), basal activity of ODC was normal and there was no reactive increase in activity following hepatectomy. In contrast, mice with "late" (3% of body weight) tumors had significantly lower basal ODC activities and the increase in ODC activity following hepatectomy was prolonged and exaggerated. We concluded that tumor burden is associated with abnormal ODC activity and that these differences are exaggerated after hepatectomy. Furthermore, although average ODC concentrations in tumor-bearing mice fell precipitously, the circadian rhythm in hepatic ODC persisted. This finding indicates early recognition by the host of tumor presence, which has a profound negative regulatory effect on hepatic ODC. Apparently, this effect does not impinge on circadian control mechanisms, indicating that these signals act independently.

A potent bombesin receptor antagonist inhibits bombesin-stimulated growth of mouse colon cancer cells in vitro: absence of autocrine effects.

Bombesin (BBS) exerts significant effects on the growth of a mouse colon cancer cell line (MC-26) in vitro. The presence of specific binding sites on MC-26 cells for gastrin-releasing peptide (GRP)/BBS-related peptides was recently reported by us. In the present study, we determined that the transcript size of the mRNA species that codes for GRP receptors is 9 kilobase pairs, which is similar to that reported for mouse Swiss 3T3 cells, using the complementary DNA probe for the GRP receptor gene from mouse Swiss 3T3 cells. We next examined the effects of potent GRP receptor antagonists, D-Phe6, bombesin(6-13)-propylamide (D-Phe6,BN(6-13)PA) and Leu13-psi-(CH2NH)Leu14-bombesin (LL-BBS), on BBS-stimulated growth of MC-26 cells in vitro. A possible autocrine role of GRP in the growth of MC-26 cells was also investigated. MC-26 cells were inoculated s.c. into male BALB/c mice, and tumors were harvested 21-28 days postinoculation. Both D-Phe6,BN(6-13)PA and LL-BBS significantly inhibited the binding of 125I-GRP to MC-26 tumor membranes in a dose-dependent manner, with 50% inhibitory concentrations of 4.5 +/- 0.52 nM and 87 +/- 6 nM, respectively. D-Phe6,BN(6-13)PA similarly inhibited the specific binding of 125I-GRP, cross-linked to a approximately 80 kilodalton binding protein on the MC-26 tumor membranes. In order to determine whether the BBS receptor antagonist, D-Phe6,BN(6-13)PA, functioned as an antagonist or an agonist of biological functions, we measured the bioefficacy of D-Phe6,BN(6-13)PA.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of colostomy on the circadian rhythm in DNA synthesis in the rat colon.

Synthesis of DNA and mitosis in gut epithelium are not constant or random events but rather are characterized by circadian rhythmicity, which we reported persists even in fasted rats. Others suggest that rhythms persist because rats anticipate food, causing nerve impulses to propagate caudally in the gut at usual meal times, or that digestive products from previous feedings cause rhythms in the lower tract. We studied colonic DNA synthesis in rats that had been given colostomies. In one study, the distal colon was isolated neurally from proximal gut by means of an end colostomy. In a second study, rats were subjected to loop colostomy; some intrinsic innervation of the gut wall remained intact. Sprague-Dawley male rats, 8 weeks old, were acclimated to a 12:12 light-dark cycle. Colostomies were performed after a 48-h fast. The rats were fed ad libitum for 4 weeks after surgery. Operated rats and an equal number (n = 30) of control rats (unoperated) were divided into four subgroups that were killed at 07:00, 13:00, 19:00, and 01:00 h. Each rat was injected with tritiated thymidine 30 min before it was killed. Proximal and distal colon were analyzed for incorporation of radioactivity (DNA synthesis). Results are reported as counts per minute per microgram of DNA and were analyzed using analysis of variance and the t test. Significant daily variation was found in proximal colon, both from control and operated rats. Rhythms were still present in colon distal to loop colostomy but were lost in the distal stump in rats that received an end colostomy.(ABSTRACT TRUNCATED AT 250 WORDS)

Circadian rhythm of cellular proliferation in the human rectal mucosa.

Circadian rhythms of DNA synthesis and cellular proliferation in the gastrointestinal mucosa have been well documented in animal models. This investigation was designed to determine whether similar rhythms could be demonstrated in the human rectal epithelium: 24 studies were performed in 16 healthy men under fasting (n = 14) and fed (n = 10) conditions. Rectal mucosal biopsy specimens were obtained through a proctoscope every 2 or 3 hours for a 24-hour span. Ex vivo measurements of tritiated thymidine incorporation into DNA were made on the mucosal samples. Feeding and time of day were each found to have an effect on the rate of thymidine incorporation into the DNA of rectal mucosal cells. Both fasted and fed subjects showed significant circadian rhythms in thymidine incorporation, which peaked at about 7 AM. Fasting lowered the overall mean thymidine uptake without altering the rhythm. Thymidine uptake generally reflects the amount of DNA synthesis in the sampled tissue. Therefore, these data may be important in the design of cancer chemotherapeutic regimens that use drugs specifically active during DNA synthesis.

Pentagastrin-stimulated DNA synthesis in mouse gut is influenced by the circadian system.

Gastrin is trophic for rodent gut mucosa. Proglumide, a competitive inhibitor of gastrin, can exert an antitrophic effect and can block pentagastrin-stimulated DNA synthesis. We have examined the influence of the circadian system on pentagastrin-stimulated DNA synthesis in the murine stomach (glandular and nonglandular stomach) and colon. We studied 224 male CD2F1 mice divided into four groups. Group A was ad lib fed (controls). Groups B, C, and D received 6-9 intraperitoneal injections of either NaCl, pentagastrin or pentagastrin + proglumide, at 8-h intervals prior to sacrifice. Mice from each group (A-D) were killed (by cervical dislocation) at 3-h intervals for 24 h. Incorporation of tritiated thymidine (DNA synthesis) was measured, and significant (p less than 0.001) circadian rhythms were found, which were not eliminated after treatment with either pentagastrin or pentagastrin + proglumide. DNA synthesis in the glandular stomach increased significantly after treatment with pentagastrin , but only during the span of time when DNA synthesis was increasing also in control mice; it had no effect at other times. Proglumide blocked the effect of pentagastrin only during the time of increasing DNA synthesis; it had no effect at other times. The identical regimen given at different times in the circadian cycle yielded significantly different results. In the intact animal, studies on the effects of various stimulators or inhibitors of DNA synthesis should be time-qualified.

Twenty-four-hour variations in ornithine decarboxylase and acid phosphatase in mice.

Polyamines are essential for cell growth and differentiation. Ornithine decarboxylase (ODC) is the rate-limiting enzyme in polyamine biosynthesis. Acid phosphatases (AP) are lysosomal enzymes that are important in normal intracellular metabolism. Twenty-four-hour variations in these enzymes may be important in understanding the temporal responses of different tissues to various stimuli. The purpose of this study was to examine a variety of tissues for fluctuations in the levels of ODC and AP over a 24-hr period. Significant circadian variations in the amount of ODC activity were observed in all tissues examined. Activity of AP varied with time of day in the liver, kidney, and heart. The highest and lowest measurements of ODC activity were as follows: liver, 81.5 +/- 7.0, 47.9 +/- 4.4; colon, 11.7 +/- 1.2, 3.1 +/- 0.7; stomach 3.1 +/- 0.4, 0.9 +/- 0.1; kidney, 420.9 +/- 0.9, 67.5 +/- 0.8; and heart, 4.7 +/- 1.0, 2.5 +/- 0.2. The highest and lowest measurements of AP activity were as follows: liver 3.8 +/- 0.1, 2.8 +/- 0.1; kidney, 3.4 +/- 0.1, 1.9 +/- 0.1; and heart, 2.6 +/- 0.1, 2.0 +/- 0.1. These findings suggest that rhythmic fluctuations in polyamine biosynthesis and lysosomal enzymes may influence other metabolic pathways differentially throughout 24 hr.

Photoperiod influences the growth of colon cancer in mice.

We have developed a mouse colon adenocarcinoma cell line that produces tumors in a dose-dependent manner when injected subcutaneously. Our previous work has demonstrated its sequential pattern of tumor area and weight under 12L:12D (12 hours light, 12 hours darkness) photoperiod. This study investigated whether shorter (6L:18D) or longer (18L:6D) photoperiods alter tumor growth. Significantly greater tumor area, weight, and group mortality were found in mice exposed to 12L:12D photoperiods as compared to either 6L:18D or 18L:6D photoperiods, and difluoromethylornithine (DFMO) was a more effective inhibitor of tumor growth under the 6L:18D photoperiod compared to 12L:12D. These results demonstrate an important role of photoperiod on tumor growth.

DNA synthetic activity in tumor-bearing mice.

The rate of DNA synthesis in normal tissues exhibits circadian rhythmicity. However, there have been conflicting reports of the effects of tumor burden on the circadian rhythm of DNA synthesis in non-cancer tissues. We have developed a mouse colon cancer (MC-26) that exhibits different growth under different photoperiods. The purpose of this study was to analyze DNA synthetic activity in tissues removed from tumor-bearing and tumor-free mice maintained under two different photoperiods. Two groups each of approximately 80 male Balb/c mice were acclimated to one of two light-dark cycles, 12L:12D or 6L:18D. Half of each group were injected with 5.0 x 10(4) MC-26 cells. Twenty-two days later, all mice were killed in subgroups at 4-6 hr intervals over one 24-hr period. Colons and tumors were removed for measurement of DNA synthesis. Results were analyzed by means of one-way analysis of variance (ANOVA) in order to determine whether DNA synthesis varied significantly within groups over the 24-hr period. The DNA synthetic activity, as measured by uptake of tritiated thymidine, exhibited significant temporal variation in the colons of control (tumor-free) mice under both the 12L:12D and 6L:18D photoperiods. The colons of tumor-bearing mice failed to exhibit a fluctuation under a 12L:12D photoperiod but did show a significant 24-hr rhythm under the 6L:18D photoperiod. The subcutaneously growing cancers did not exhibit a circadian variation in DNA synthetic activity under either photoperiod. Both photoperiod and the presence of cancer appear to affect the DNA synthetic activity observed in mice bearing the MC-26 colon cancer.

Restricted feeding schedules alter the circadian rhythms of serum insulin and gastric inhibitory polypeptide.

Insulin and gastric inhibitory polypeptide (GIP) have a circadian rhythm of secretion that is altered by various feeding schedules. We acclimated rats over 3 weeks to one of 6 different feeding schedules. They were then killed at intervals over one feeding cycle. Blood was collected, and their stomachs were weighed. Hormones in the serum were measured by radioimmunoassay. When highest and lowest measured concentrations were compared in ad libitum fed rats, insulin more than doubled (445 +/- 50 to 993 +/- 180 pg/ml) and GIP more than tripled (682 +/- 108 to 1964 +/- 145 pg/ml) during a 24-h period. With restricted schedules, concentrations correlated with the feeding schedule, not the light-dark cycle. Hormone levels rose higher during feeding and fell lower with fasting than in ad lib fed rats. For example, GIP in one study fluctuated from 468 +/- 22 to 6433 +/- 432 pg/ml. In another example, insulin ranged from 30 +/- 5 to 2259 +/- 406 pg/ml during a 24-h period. However, insulin did not always correlate well with stomach weight. Circadian rhythms occurred for insulin with all feeding schedules and for GIP with all schedules except fasted rats. This finding implies an endogenous insulin rhythm, whereas food intake controls GIP secretion. Thus, disruption of normal circadian cycles of feeding may yield misleading information about gut hormone secretion.

Circadian rhythms in gastrin receptors in rat fundic stomach.

Circadian rhythmicity in the number of gastrin receptors in rat fundic mucosa was characterized and was related to the concentrations of gastrin in serum and in antrum. Male Sprague-Dawley rats were acclimated to 12 hr light alternating with 12 hr darkness. Subgroups of six rats each were killed at 4-hr intervals. Fundic mucosa was collected for measurement of gastrin receptors; serum and antral tissues were collected for measurement of gastrin levels by radioimmunoassay. Circadian periodicity in the data was determined by cosinor analyses. In both freely fed and fasted rats, gastrin receptors showed circadian variation (range 2.5-10 fmol/mg protein), as did serum gastrin concentrations (range in fed rats 195-407 pg/ml). The phasing of the intrinsic circadian variation in gastrin receptor level that was observed in the fasted rats was advanced by a few hours in fed rats. This shift is probably due to food-induced gastrin release, resulting in gastrin-mediated down-regulation of gastrin receptors, followed by up-regulation of gastrin receptors. Food-related effects were thus superimposed upon the intrinsic circadian rhythms in gastrin receptor levels, causing the circadian variation in gastrin receptor levels in the fed rats to be shifted forward compared to that in the fasted rats. No significant circadian rhythms, on the other hand, were found in concentrations of gastrin in the antrum. These results suggest that changes in sensitivity of target tissues to hormones are related to both intrinsic circadian rhythms in levels of hormone receptors and also to food-related changes in hormone-receptor levels mediated by changing serum hormone levels.

Daily variations in the formation of gastric ulcers caused by cervical cord transection in the rat.

Cervical spinal cord transection is used as a model of mammalian stress ulcerogenesis. Circadian variations in gastric ulceration have been demonstrated in other animal models. We investigated whether gastric ulceration changed after cervical cord transection throughout a 24-h period by subjecting different groups of rats to cord transection. Ulcers were quantitated using an index described by Szabo and colleagues. Ulcer formation showed significant variation as a function of time of lesioning, with least severe ulceration at 8 AM. We found that cord transection, like other models of gastric ulceration, is also influenced by the circadian system.

Several cytokinetic methods for showing circadian variation in normal murine tissue and in a tumor.

Circadian rhythmicity in cell proliferation was studied by several cytokinetic techniques in 8-week-old CD2F1 and Swiss male mice. In separate experiments on four different dates, subgroups of six or seven mice (fed ad libitum and standardized to 8 hours light alternating with 16 hours of darkness) were killed at 3-hour intervals over a period of 24-48 hours. Cosinor analyses were used to determine the parameters of circadian periodicity in the data. Flow cytometric (FCM) analyses of DNA synthesis in tongue and stomach were compared with estimates of tritiated thymidine (3HTdR) incorporation into the DNA in these tissues. Circadian variation in tongue epithelium was reproducible in phasing and in range-of-change (100-140%) in seven 24-hour studies. The proportion of cells in G1 and G2 in tongue epithelium also demonstrated a circadian periodicity. When DNA synthesis in stomach was analyzed by the two methods, the results were entirely different and thus not comparable. The circadian rhythms in 3HTdR uptake agreed with those reported previously, but results of analyses by FCM were less certain, since epithelium of this organ could not be analyzed separately. Circadian rhythms were detected by FCM in G2, S, and G2M in Ehrlich ascites carcinoma (EAC). The mitotic index of this tumor varied with a circadian periodicity similar to that of the FCM-derived rhythm in G2M. This study validates the reliability of older techniques, such as 3HTdR uptake and mitotic indices, and suggests that circadian rhythms can be important in studies of cellular properties using FCM.

A sample-grouping technique for paraffin embedments.

A technique is described which facilitates histological preparation of multiple tissue specimens for light microscopy. The procedure enables the investigator to separate and label identifiable subgroups from a larger number of specimens in one histological section. After standard fixation, murine esophagi were arranged longitudinally and secured within segments of murine intestine. Markers such as plant fibers and human hairs were threaded alongside the esophagi within each intestinal casing. After standard dehydration and infiltration, several segments of intestine were arranged parallel to each other and at right angles to the intended plane of sectioning and were embedded together in one paraffin block. This method made it possible to assemble onto one microscope slide cross sections of 42 individual esophagi in 6 identifiable subgroups, each containing 7 esophagi.

Circadian rhythms in phases of the cell cycle in the hamster as demonstrated by flow cytometry.

Circadian rhythmicity in the phases of the cell cycle of several epithelial tissues of the hamster was analysed by flow cytometry. Hamsters were killed every 3 hr for 24 hr to permit observation of the effect of 'time of day' on the proportion of cells in the various phases of the cell cycle. Bone marrow and epithelium from cheek pouch, oesophagus and tongue were isolated and processed to single cell suspensions for analysis. The only systematic difference in the collection of the data was the time of day when the hamsters were killed. From the resulting DNA histograms, derived by flow cytometry (which was chosen as the technique for this study because of demonstrated applicability in determining cellular properties), the G1, S, and G2 fractions were estimated. Multiple linear regression techniques were used to estimate circadian periodicity in the data. The calculated circadian rhythms in tongue and oesophagus, derived by flow cytometry, were consistent with murine rhythms previously reported, as derived by other techniques. Circadian rhythms also were demonstrated by flow cytometry to be present in the cheek pouch epithelium, as has been reported by others. Although there was considerable fluctuation in findings from the bone marrow, the fluctuation was not of circadian periodicity, perhaps because of the mixed cell population. This study validates the reliability of older techniques, such as mitotic indices, labelling indices, and uptake of tritiated thymidine, which show the rhythmic nature of cell division in vivo.

Survival and cell cycle kinetics responses of Chinese hamster ovary cells, and clones of human adenocarcinoma of the stomach and astrocytoma to diaziquone (AZQ) in vitro.

The anticancer agent 1,4-cyclohexadiene-1,4-dicarbonic acid, 2,5-bis(aziridinyl)-3,6-dioxo-, diethyl ester (AZQ) (NSC 182986) was studied in vitro to determine survival, cell cycle stage sensitivity, and cell cycle kinetics effects. One hour treatments with AZQ doses ranging from 1 micrograms/ml to 25 micrograms/ml revealed that human stomach tumor clones were most sensitive of three cell types studied to to the killing effects of AZQ; this sensitivity was followed in order by human astrocytomas and Chinese hamster ovary (CHO) cells. Depending on the AZQ dose, nondividing CHO cells were 10 to 180 times more sensitive than dividing CHO cells. Synchronized CHO cells were most sensitive to AZQ's killing effects when treated at the late S/G2 phase boundary, with the overall order of sensitivity being late S/G2, G2, mid-S, and G1 phase. Mitotic cells were neither killed by doses used in these studies, nor were they inhibited in their progression from mitosis into the G1 phase. Synchronized CHO cells treated in all other phases of the cell cycle were either blocked completely or delayed for up to 2 hours in their progression through the cell cycle. Flow microfluorometry (FMF) studies on exponentially growing CHO cells demonstrated that even at noncytotoxic doses (1 microgram/ml), AZQ caused very large, but reversible enrichments of cells in the S and G2 phases of the cell cycle. Since AZQ has already been shown to be effective against a variety of animal and human tumors (especially brain tumors) the data reported here may be useful in designing more effective treatment schedules and drug combination regimens.

In vitro cellular characteristics and survival responses of human astrocytoma clones to chloroethyl-nitrosoureas and dianhydrogalactitol.

Three permanent clones were derived from a single astrocytoma cell line and were characterized for in vitro cell kinetics, chromosomal properties and for their responses to the anticancer drugs: 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU); 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea (MeCCNU); 1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl-1-nitrosourea) (PCNU); and 1,2:5,6-dianhydrogalactitol (GAL); all of which have been shown to cross the blood brain barrier. The clones showed different population doubling times, saturation densities, plating efficiencies, chromosome counts, ploidy, cell cycle phase distributions and DNA indices. The only positive correlation among these parameters was between the population doubling times and the modal chromosome numbers; the lower the chromosome number, the shorter the doubling time. No correlation was observable between any of the cellular properties and responses to the four drugs. The clones showed a differential sensitivity to the nitrosoureas, seen maximally as a 600-fold difference in survival between two of the clones treated with the same dose of BCNU. In contrast, the clones exhibited almost identical and uniform sensitivities to galactitol, suggesting that this agent exerted its cytotoxic effects by similar mechanisms in each of the clones. By comparison BCNU (at the tested doses and duration of drug exposure used in this study) was found to be the most effective of the agents tested.

Circadian stage dependence in radiation: response of dividing cells in vivo.

1) When the mouse corneal epithelium wa irradiated with one dose or ionizing radiation, the effect on mitosis varied depending upon the time in the 24-h period when it was irradiated. 2) Release of the radiation-induced mitotic block, as measured by appearance of a recovery wave of mitotic cells, occurred only during the daily increase in mitotic index in the control animals. 3) The entire wave of recovery occurred within the same time as the controls, and no rebound was seen. 4) Mitotic delay was dose-dependent. 5) The results of these experiments emphasize the importance of considering the circadian system when studying cell division in vivo.