DNA Synthesis Index Research Articles

DNA Synthesis Index: Higher for Human Gallbladders With Cholesterol Gallstones Than With Pigment Gallstones<xref ref-type="fn" rid="FN2">2</xref><xref ref-type="fn" rid="FN3">3</xref>

[3H]dThd uptake by the gallbladder epithelium was estimated in 33 patients with cholesterol stones, in 13 patients with pigment stones, and in 12 gallbladders without stones. Proliferative parameters were estimated by autoradiography after in vitro incubation with [3H]-dThd. Stones were identified by quantitative infrared spectroscopy. The degree of inflammation of the gallbladder wall was estimated by a histologic scoring method. In the gallbladders containing cholesterol stones the DNA synthesis index (1.39 +/- 0.28%) was higher (P less than .01) than in the gallbladders without stones (0.19 +/- 0.04%). No significant increase in proliferative parameters was found in the gallbladders with pigment stones (0.24 +/- 0.06%). No correlation was found between total stone number, weight or volume, and the DNA synthesis index. No evidence was observed that inflammation could influence the epithelial cell proliferation. Something in the bile of patients with cholesterol stones rather than the physical presence of stones may be the cause of the variations observed.

Normal mitotic reactivity of fibroblasts and mesothelial cells in thrombocytopenic rat.

Mitotic reactivity following 48/80-induced mast-cell secretion was studied in the mesentery of rats made thrombocytopenic, 7 days following a single injection of melphalan. In spite of a low platelet count (7% of normal), the mitogenic reaction of the mesenteric fibroblasts and mesothelial cells was normal as judged by DNA-synthesis and mitotic index. The findings suggest that platelets and platelet-growth factors are not essential for the mast-cell-mediated mitogenic reaction of these two types of connective-tissue cells studied in vivo.

IMMUNOLOGICAL CHALLENGE CAUSES MITOGENIC STIMULATION IN NORMAL CONNECTIVE TISSUE CELLS

Because polyamine-induced mast-cell secretion causes mitogenesis in surrounding tissue cells in the rat mesentery, we studied whether an immunological challenge in guinea-pig and rat that normally involves mast-cell secretion caused mitogenesis in adjacent normal tissue cells. The appropriate antigen was injected intraperitoneally into pre-sensitized animals. Mast-cell secretion was assessed as histamine release and proliferation by estimating DNA synthesis and mitotic index in the mesentery of the intact animals. In both species the immunological challenge causing significant release of mast-cell histamine was followed by a marked mitogenic response in fibroblasts and mesothelial cells of the mesentery. Although the present study was not primarily designed to study the molecular mechanisms for stimulating cell proliferation, the results suggest that the role of primary mast-cell products on a quantitative basis is significantly different in the mitogenesis following immunological challenge than in polyamine-induced mast-cell mediated mitogenesis in the rat.

Effect of naloxone and D-Met 2-Pro 5-enkephalinamide treatment on the DNA synthesis in the developing rat brain

Effects of a single dose of naloxone and of D-Met 2-Pro 5-enkephalinamide on the DNA synthesis in the forebrain, hypothalamus and cerebellum of 11 day old female rats were studied. As an index of DNA synthesis the rate of incorporation of 3H-thymidine into DNA was measured 30 min after a sc. injection of 40 μCi/100 g b.w.. A time dependent effect of naloxone administration on cerebral DNA synthesis was observed. In the forebrain at 1 and 3 hrs after naloxone injection an increased rate of 3H-thy-midine incorporation into DNA was found followed by a marked decrease at 9 and 12 hrs. The effect in the hypothalamus was similar but the initial increase at 1 hr was absent. On cerebellar DNA synthesis naloxone had no effect. The administration of D-Met 2-Pro 5-enkephalinamide resulted in a marked reduction in the labelling of cerebral and hypothalamic DNA between 1 to 12 hrs. Except a decrease at 1 hr no effect was found in the cerebellum.

Evidence for cell division in synoviocytes in acutely inflamed rabbit joints.

DNA synthesis has been measured both by Feulgen cytophotometry, quantified by the DNA synthesis index, and by tritiated thymidine autoradiography, quantified by the labelling index. In the early acute inflammation resulting from the intra-articular challenge of ovalbumin in sensitised rabbits both indices rose considerably, so that at least 1 in 10 synoviocytes was heavily labelled 3 days after challenge. The results are compatible with the concept that even such apparently differentiated synoviocytes are capable of cell division.

Rapid onset of vascular wall protein synthesis with increase in lability of blood pressure in rabbits.

1. Structural and functional changes resulting from baroreceptor denervation were studied in rabbits 4 days after surgery. 2. After section of the aortic and carotid sinus nerves, arterial pressure was characteristically labile although the mean pressures were not significantly different from those of control animals. 3. The responsiveness of isolated ear arteries and cephalic veins from deafferented animals to noradrenaline and electrical stimulation of their sympathetic innervation was similar in both groups. 4. Neuronal uptake of noradrenaline into arterial tissue was similar in both groups, although uptake into the cephalic veins of deafferented animals was less than in the control group. 5. [3H]Thymidine uptake, an index of DNA synthesis, was similar in both groups, radioautography revealing no difference in smooth muscle cell labelling. 6. Uptake of [3H]proline, an indicator of protein synthesis, by aorta, ear, brachial, basilar and renal arteries from deafferented animals was greater than in control animals. 7. Baroreceptor denervation altered protein metabolism in the arterial wall.

DNA synthesis in human rheumatoid and nonrheumatoid synovial lining.

The DNA content per nucleus has been measured in the lining cells of 12 specimens of rheumatoid and 12 of nonrheumatoid synovial tissue. Optimal conditions for this reaction on these cells have been defined, and an index of DNA synthesis has been applied to give a quantitative measure of the degree of synthesis. This has given evidence of some DNA synthesis in both types of synoviocytes, compatible with the amount found in a slowly self-replacing tissue. There was no difference in the amount of synthesis in the rheumatoid and nonrheumatoid synoviocytes.

Testosterone action on BFU-E and CFU-E: Correlation between enhancement of proliferative rate and amplification of pool size

As previously reported, a single administration of testosterone propionate (TP) in ex-hypoxic polycythemic mice induces an 18–24 hr amplification of the erythroid burst-forming unit (BFU-E) pool and a 60-hr expansion of the erythroid colony-forming unit (CFU-E) compartment. Both phenomena are here shown to be temporally associated with an increase of the in vitro 3H-TdR sensitivity of these compartments, thus indicating an elevation of their proliferative rate. On the other hand, no significant modification of both the DNA synthesis index and the pool size of BFU-E and CFU-E were observed at respectively 60 or 18 hr. At either time interval, both 3H-TdR sensitivity and compartment size were not modified at the level of the myeloid-macrophage colony-forming unit (CFU-C). It is therefore suggested that the early and late expansion of respectively BFU-E and CFU-E number after TP injection is at least partially mediated by enhancement of the proliferative rate within the respective compartments. Finally, mechanisms underlying TP action on BFU-E and CFU-E pools are discussed in the light of both present and previous observations.

Characterization of Feline Whole-blood Cultures and Determination of the Frequency of Radiation-induced Dicentrics in Human and Feline Lymphocytes

The Ham's F-10 and PHA culture system was applied to whole feline blood and cell-cycle characteristics such as DNA synthesis and mitotic indices were studied. The results are comparable to those obtained from human whole-blood cultures. The yields of dicentrics were also determined in lymphocytes from X-irradiated human and feline blood. The ratio between the experimental yields of dicentrics in human as compared to feline lymphocytes was 1:0.27.

Neurotrophic Contribution to a Proposed Tripartite Control of the Mitotic Cycle in the Regeneration Blastema of the Newt,Notophthalmus (Triturus) viridescens

My work has shown that the neural dependence of the limb regenerate (blastema) for growth, is retained in vitro and it follows the normal in vivo pattern. Implanted dorsal root ganglia promote localized growth in cultured blastemata whereas in the absence of nerves little or no development ensues. Our transfilter studies show that the neurotrophic effect, which appears to be mediated by a factor capable of transmission through thin filters of low porosity, is manifested in a proximo-distal gradient of mitotic activity which diminishes with distance from the nerve source. This mitogenic effect appears to require at least 48 hr for expression, which corresponds to one revolution of the cell cycle. In the absence of nerves, both DNA synthesis and mitotic index in the blastema decline significantly; however, a basal level is maintained which may be nerve independent. In this context and in the light of pertinent recent literature, we present a model in which nerves, the apical epidermis and insulin (tripartite control) all play essential roles in the mitotic cycle.

Studies on follicular growth in the immature rat and hamster: Effect of a single injection of gonadotropin or estrogen on the rate of3H-thymidine incorporation into ovarian DNAin vitro

Initiation of follicular growth by specific hormonal stimuli in ovaries of immature rats and hamsters was studied by determining the rate of incorporation of3H-thymidine into ovarian DNAin vitro. Incorporation was considered as an index of DNA synthesis and cell multiplication.

In vitro sensitivity test for antineoplastic agents with an index of DNA synthesis of tumor cells

In vitro sensitivity test for antineoplastic agents with an index of DNA synthesis of tumor cells

Endogenous hypergastrinemia and cell proliferation in the fundic mucosa in dogs.

Endogenous hypergastrinemia (P less than 0.01) was produced in 4 dogs with a Heidenhain pouch through surgical exclusion of the pyloric antrum. This was followed by acid hypersecrection and increased DNA synthesis and mitotic activity (P less than 0.01) in the mucosa of the Heidenhain pouch. The acid hypersecretion induced by histamine in beeswax in 4 other dogs did not increase the proliferative parameters in the pouch mucosa of the latter. A positive correlation (r = 0.84, P less than 0.01) was found between the serum gastrin levels and the DNA synthesis index values measured in the 4 animals before antral exclusion, after antral exclusion, and after antrectomy. These data support the hypothesis that the trophic effect of gastrin on the fundic mucosa can be elicited by physiological doses of endogenous hormone.

Fractional incorporation of [3H]thymidine and DNA specific activity as assays of inhibition of tumour growth.

The Fractional Incorporation (FI) of [3H] thymidine ([3H]TdR) has been examined in small lung tumours after cyclophosphamide (CY) treatment in vivo and compared to the DNA specific activity (SA) at different times after treatment. FI was found to correlate with the incidence of labelled cells after treatment, whereas SA did not, due to the loss of DNA from drug-killed cells 72 h after treatment. The FI is independent of the precursor concentration in the tissue, and therefore may give a better index of DNA synthesis in irregularly perfused tissues than SA. Following either CY or 60Co radiation treatment, the time necessary for FI to reach the pretreatment level is quite similar to the growth delay measured for the FI depression 45 h after treatment and growth delay has been established in the Lewis lung tumour, which would allow the prediction of growth delay induced by another agent to be made within 2 days of treatment.

Importance of progesterone in DNA synthesis of pregnancy‐dependent mammary tumors in mice

Hormone responsiveness of pregnancy-independent mammary tumors in SHN mice and pregnancy-dependent tumors in GR/A mice and in F1-hybrids between these strains was studied. Force-bred female mice with palpable mammary tumors were given subcutaneous injections of several hormones singly or in combination twice daily from 1 day before to 1 day after parturition. One group received a graft of three pituitary glands under the kidney capsule (3AP) during days 12-14 of pregnancy. One day after parturition, the in vivo incorporation of 3H-thymidine into DNA of normal and neoplastic mammary glands was determined as the index of DNA synthesis. Plasma prolactin in some groups of GR/A mice was assayed by radioimmunoassay. In GR/A mice, estradiol benzoate (EB: 0.5 mug X 2/day) or 3AP had no effect on either normal glands or tumors, despite an increase in plasma prolactin level. Progesterone (P:100 or 1,000 mug X 2/day) significantly increased DNA synthesis of both normal and neoplastic glands when compared to the controls, while the plasma prolactin level in this group was low. The administration of human placental lactogen (HPL: 100 mug X 2/day) stimulated DNA synthesis of normal glands only. P plus HPL promoted DNA synthesis of tumors more than P alone, but not P plus EB. While DNA synthesis of the tumors of SHN was never affected by these hormone treatments, the hormone responsiveness of tumors of F1-hybrids was almost the same as that of GR/A mice; the effect of P was prominent. Correlation of DNA synthesis between pregnancy-dependent mammary tumors and normal glands was significant only in groups treated with P alone or in combination with other hormones.

Effects of Pituitary Graft and 2-Bromo-α-ergocryptine on Mammary DNA Synthesis in Mice in Relation to Mammary Tumorigenesis2

3H-thymidine incorporation into mammary DNA as an index of mammary DNA synthesis in C3H/He virgin females was significantly higher at proestrus than at estrus and diestrus. The rise in incorporation at proestrus was completely blocked by the administration of 2-bromo-alpha-ergocryptine (Bromocriptin) 1 day before proestrus. The incorporation was maintained at a higher level than that at proestrus by pituitary grafts. The results supported the view that mammary DNA synthesis, which is stimulated by prolactin, is an important factor for mammary tumorigenesis.

The relation between cell division rate and 3 HTdR incorporation in organ culture.

AbstractRates of incorporation of labelled thymidine (RIT), radioautographic labelling index for DNA synthesis (LI) and mitotic incidence following colcemid metaphase arrest (MI) were measured in organ cultures of newborn and adult rat lung. In adult cultures these three parameters correlated well, being low after explantation and reaching a maximum after two to three days. In newborn cultures RIT fell several fold over the first 24 hours after explantation and, in this respect, did not correlate with LI and MI. The changes in RIT over the first 24 hours appear to be due to changes in the degree of competition between endogenous TdR and exogenous labelled TdR, probably caused by leakage of the intracellular thymidine pool following explantation. The report emphasizes the need to check RIT data against radioautographic evidence before accepting it as an index of DNA synthesis.

The Effect of L-Asparaginase on the Nucleic Acid Metabolism and Cell Cycle of Human Leukemia Cells

Abstract The effect of L-asparaginase on the cell cycle and nucleic acid synthesis of leukemic cells was studied in five children with acute lymphoblastic leukemia. Following an intravenous infusion of the drug, serial marrow samples were obtained for buffy coat volume, mitotic index, and autoradiographic assessment of DNA and RNA synthesis using tritiated thymidine and tritiated uridine, respectively. A rapid decline in buffy coat volume indicated a lytic effect on lymphoblasts. There was a greater kill of proliferative (blasts in the cell cycle) than nonproliferative (G0) leukemic cells. Mitotic indices changed little until 24 hr; in contrast, thymidine labeling indices decreased markedly to less than 50% of control by 6 hr. The changes in labeling indices prior to changes in mitotic indices indicated that L-asparaginase blocked the entrance of cells into the DNA synthesis period of the cell cycle. Cells already in DNA synthesis appeared to continue into mitosis. Uridine labeling indices decreased progressively in all patients. Uridine uptake was inhibited equally in both proliferative and nonproliferative blasts. Therefore, inhibition of RNA synthesis by L-asparaginase was independent of the proliferative activity of the marrow.

Developmental Physiology of Cestodes. IX. Cytological Characteristics of the Germinative Region of Hymenolepis diminuta

The germinative region of developing Hymenolepis diminuta is defined based on cytological characteristics and DNA synthetic activity as beginning 200 ,u posterior to the apex of the scolex, and extending posteriorly to the point of germinal primordia formation. The structure of the germinative type in this region is described. The kinetics of division in the germinative region are studied, and estimates of the duration of G1, S, and G2 of prophase are 3.0, 2.3, and 3.2 hr, respectively. The time for a complete mitotic division is estimated to be 8.5 + hr. Although proglottisation in most cyclophyllidean cestodes has been attributed to budding from the unsegmented region immediately posterior to the scolex (Wardle and McLeod, 1952; Roberts, 1961), no cytological definition or exact location has been given to the so-called germinative area. Roberts (1961) indirectly defined the germinative area of Hymenolepis diminuta as the area behind the scolex in which genital primordia are not observed. Sandeman (1959) defined the germinative area of Capsulata edenesis, a parasite of Limosa lapponica, as a posteriorly located diffuse area of the strobila from which the rest of the strobila originates. If the germinative region is indeed responsible for proglottid production, the cellular activity in this region should be significantly different from the rest of the strobila, and division indices and an index of cells involved in DNA synthesis could be used as criteria of definition. This region should also contain a cytologically identifiable type which could act as a stem cell for production of proglottids. Bell and Smyth (1958) and Wikgren (1964) have used the criterion of a mitotic index to estimate growth in trematodes and cestodes. The use of the incorporation of tritiated thymidine into DNA as an index of DNA synthesis and, therefore, as an index of replication was discussed by Clever (1967). Received for publication 30 July 1970. * This investigation was supported by Research Grant AI-06153 and Training Grant 5 TOI AI226 from the NIH, U. S. Public Health Service, Bethesda, Maryland 20014. tPresent address: Department of Biology, Notre Dame University, Notre Dame, Indiana 46556. In the present study we have investigated the cytological characteristics of the germinative region of H. diminuta and a possible germinative type within this region. The duration of the cycle of the proposed germinative has been estimated. MATERIALS AND METHODS Recovery and incubation of H. diminuta Male Holtzman rats (120 to 180 g) were inoculated with either 100 or 10 cysticercoids and worms were recovered 2, 4, and 6 to 14 days postinfection, respectively. Worms were recovered 2 and 4 days postinfection from the hosts' small intestine by scraping the intestinal mucosa with a glass microscope slide into phosphate-buffered (pH 7.4) balanced salt solution (BSS) (Roberts and Fairbairn, 1965) or into tris-maleate-buffered Ringer's solution (Read et al., 1963) with 0.1% added glucose (TMR + G; pH 7.4). The gut scrapings were examined under a dissecting microscope, and the worms were separated from the debris with a Pasteur pipette. Older worms, 6 to 14 days postinfection, were flushed from the rat small intestine with a small amount of salt solution. The worms were washed, incubated 15 min in BSS or TMR + G containing 0.4 mg streptomycin and 500 units potassium penicillin-G per ml, and then incubated in the salt solution containing an experimental substrate. All incubations were done in 25-ml Erlenmeyer flasks with a tissue-to-medium ratio of 60 mg worm fresh weight/ml incubation fluid. Incubations were at 37 C in an atmosphere of N2:CO2 (95:5). Following the incubation, the worms were washed with salt solution and fixed for cytological studies. Preparation for determination of mitotic index Worms were incubated in a concentration of colchicine for a length of time necessary to obtain the maximum number of arrested metaphase figures. Entire 2and 4-day-old worms and the ant rior 2 cm of 6to 14-day-old worms were fixed immediately in 3:1 (v:v) 70% ethanol:neutral

INFECTIOUS MONONUCLEOSIS, AN AUTORADIOGRAPHIC STUDY OF DNA- AND RNA-SYNTHESIS.

The in vitro DNA and RNA synthesis of infectious mononucleosis (I.M.) cells have been studied in four patients. For evaluation, monocytoid, lymphocytoid and plasmocytoid I.M. cells were distinguished. Short‐term incubation with H3‐thymidine revealed that the DNA synthesis index was highest in plasmocytoid forms with 21 per cent labelling, followed by monocytoid I.M. cells with 7.5 per cent and lymphocytoid I.M. cells with 1.3 per cent uptake. In vitro RNA synthesis was also characterized by most active rates of incorporation of H3‐uridine and H3‐cytidine in plasmocytoid cell types and a similar drop in the intensity of uptake for the other two forms. The still enigmatic origin and function of these irritation cells of the peripheral blood are emphasized. The rather striking autoradiographic findings of cellular proliferation and active RNA synthesis in these cells reveals properties of immature cells. The previously suggested possibility of intracellular viral synthesis is considered to be rather unlikely from our findings. On the other hand, a relationship to multipotent mononuclear stem cells which today have to be assumed to occur in the peripheral blood of even healthy individuals, is discussed.