Tritiated Thymidine Studies Research Articles

Müller cells express the neuronal progenitor cell marker nestin in both differentiated and undifferentiated human foetal retina.

Tritiated thymidine studies suggest that Müller cells are the last cells born in the retina, although several authors describe Müller cells throughout the retina from very early ages. In this study immunohistochemistry was used to identify progenitor and Müller cells in human foetal retina. Antibodies to nestin (an intermediate filament protein expressed by neural progenitor cells), vimentin, cellular retinaldehyde binding protein (CRALBP) and glutamate and aspartate transporter (GLAST), which are each expressed by Müller cells, were used in combination with anti-Ki67 to identify proliferating cells. By definition, Ki67-positive proliferating cells were present in undifferentiated retina, but not in differentiated retina. Nestin-immunoreactive (IR) cells colocalized with vimentin throughout the retina. CRALBP-IR was detected in differentiated retina and in some proliferating cells. GLAST-IR cells were present only within the differentiated region. Nestin, vimentin and CRALBP each colocalized with mitotic Ki67-IR cells, suggesting that in foetal retina Müller cells and retinal progenitor cells are overlapping populations and that Müller cells are end-stage progenitor cells.

5‐Fluorouracil–induced small bowel toxicity in patients with colorectal carcinoma

The report by Fata et al.1 and the historical commentary by Kennedy2 document the well-known toxicity associated with fluoropyrimidine therapy, a diarrheal syndrome that may be life-threatening and can lead to death. Fata et al. indicate that the anatomic lesions in the cases reported were located in the small intestine, implying that this is not the usual site of the injury and that the mechanism of 5-fluorouracil (5-FU)–induced injury is related to vasospasm. With regard to the anatomic localization of a lesion in the small intestine, it is likely that this is, in fact, the most common site of 5-FU–induced intestinal ulceration and secondary diarrhea. Tritiated thymidine studies of the intestinal tract indicate that the proliferative activity of the cells in the small intestine is higher than in the large intestine and the movement of the crypt cells to the tip of the villus occurs over a shorter time interval for the small versus the large intestine. Therefore, the glandular cells of the small intestine would at least conceptually be more sensitive to the cell cycle specific action of the fluoropyrimidine antimetabolites. Furthermore, a 5-FU–induced lesion in the large intestine has not been specifically described in the literature, although Kennedy refers to a “pseudomembranous enterocolitis” from a 1959 report and suggests that the entire gastrointestinal tract may be subjected to mucosal damage by 5-FU. Although the diarrheal syndrome associated with the fluoropyrimidines has not been studied prospectively and has not been routinely evaluated with intestinal assessment with radiologic or endoscopic investigations, a previously reported case study by our group did demonstrate that floxuridine infusion induced radiographic lesions in the small intestine not unlike those seen with Crohn disease3 and was reversible with drug withdrawal. These cases were similar to those described by Fata et al. Additional aspects of the fluoropyrimidine-induced intestinal syndrome relate to the specific fluoropyrimidine used, the drug schedule of administration, and the concomitant use of leucovorin for biomodulation. Floxuridine (5FUdR) is more commonly associated with diarrhea than 5-FU for unknown reasons, although the pharmacokinetic and pharmacodynamic differences between the two fluoropyrimidine analogues is well established. In terms of the drug schedule, 5-FU administered by protracted venous infusion (PVI) is uncommonly, if only rarely, associated with diarrhea. On the other hand, bolus 5-FU results in diarrhea as a major gastrointestinal toxicity. These clinical observations suggest that infusional 5-FU may be mechanistically or pharmacologically different from bolus 5-FU in terms of cellular effect, as suggested by Sobrero and Bertino,4 in which bolus 5-FU effects RNA and infusional 5-FU effects DNA synthesis and the pattern and frequency of toxicity may therefore differ. Finally, what is the role of leucovorin modulation in inducing the intestinal lesion? Early studies from Roswell Park and subsequently by the Gastrointestinal Tumor Study Group and others5 established that leucovorin modulation and 5-FU administration resulted in a dose-limiting “cholera-like” syndrome of diarrhea and secondary dehydration. Furthermore, in the Phase I trials of infusional 5-FU with concomitant leucovorin, diarrheal toxicity was commonly observed.6 It would appear, then, that 5-FU biomodulation with leucovorin increases the frequency and severity of 5-FU–induced diarrhea (in both bolus and infusional schedules of 5-FU), but the specific mechanism is unclear. Vasospastic effects or alterations in fibrinolytic activity seem unlikely, and it is possible that leucovorin modulation of 5-FU results in an “FUdR”-like effect in the mucosal lining cells of the small intestine, leading to mucosal ulceration and resulting in diarrhea. I suspect that the diarrheal toxicity associated with 5-FU is almost always related to a small intestinal lesion and that the incidence and intensity is related to the schedule of 5-FU administration and the use or nonuse of concomitant leucovorin modulation. Jacob Lokich M.D.*, * The Cancer Center of Boston, Boston, Massachusetts

The Cell Cycle Status of Fetal Granulocytic Progenitors is Different than Adult Progenitors • 775

Septic premature infants are frequently neutropenic, a characteristic also observed in a neonatal rat model. In rats, the responsible mechanism is linked to a near-maximal proliferation of granulocyte progenitors in the non-infected state, which limits their proliferative response to sepsis (Pediatr. Res., 1982). Although this scenario may explain the frequent depletion of marrow neutrophils during sepsis in human neonates, the cycling status of their granulocytic progenitors is unknown. To test this, we performed tritiated thymidine clonogenic studies and flow cytometric DNA analysis on CD34+ cells isolated from fetal bone marrow (BM), cord blood (CB), and for controls, from adult BM. For clonogenic studies, CD34+ cells were incubated with either tritiated or non-radioactive thymidine prior to plating in methylcellulose, and colonies were scored after 14 d of culture. Cell kill, a function of thymidine uptake and an indirect measure of active cycling status, was greater for granulocytic clones in CB than in adult BM (X±SD; 49±7% vs. 25±12%; p < 0.05). To more directly determine cell cycle status, flow cytometric DNA analysis of CD34+ cells was performed using a propidium iodide method (Cycle TEST™ PLUS DNA Reagent Kit, Becton Dickinson). In the fetal BM, the proportion of cells in S-phase nearly doubled those of adult BM, while only a small proportion of CB CD34+ cells were in S-phase (*p < 0.01 vs. adult cells). These results contrast with those of the tritiated thymidine studies and suggest that thymidine uptake in CB CD34+ cells might occur in G1-phase as well as S-phase. To investigate this, studies utilizing acridine orange dye to differentiate G0 and G1 status in CD34+ cells from CB and adult BM are underway. Cell cycle status studies of granulocytic progenitors in the human fetus and neonate might further delineate the mechanism(s) associated with the neutropenia that occurs during bacterial sepsis. Table

In vitro chick pre-cardiac explant tissue differentiation during spaceflight on SpaceHab-02.

Chick precardiac tissue explants were cultured on the 8-day mission of STS-60, space shuttle Discovery. Development of in vitro cultures of precardiac chick tissue from embryo stages 5 though 8 (H-H) were initiated during orbit and were terminated after approximately fifteen hours of 37 degree C culture. Transmission electron microscopy and tritiated thymidine studies were performed postflight. No significant differences in cell proliferation were observed between flight and ground controls. Electron-microscopic studies revealed stage 8 explants were capable of differentiation during flight in a pattern which matched ground control tissues. As anticipated, stage 7 explant tissues had differentiated to a lesser extent compared to stage 8 tissues. Interestingly, stage 7 precardiac explant flight tissue differentiation was less than ground control tissue. This difference in differentiation between flight and ground cultures was enhanced in stage 6 tissues, as high levels of myofibril organization were only seen in ground controls. Other cellular components such as Golgi apparatus, junctional complexes, and mitochondria were present and appeared normal and healthy.

Arthritis Induced with Freund's Adjuvant and its Relationship to Changes in the Stomach, Small Intestine and Thymus: A Histological and Tritiated Thymidine Study on Rats

The aim of the study was to elucidate histological changes provoked by Mycobacterium butyricum toxine in the rat joints, stomach, small intestine and thymus and the uptake of tritiated thymidine. Freund's complete adjuvant was injected into the base of the vastus muscle of 35 rats; 21 rats served as controls. Fifteen rats displayed histologically mild synovitis in the ankle joint, two periostitis and three muscle infiltrates. Histological study revealed no difference between the test animals and the controls in the stomach, small intestine and thymus. The thymidine uptake of the adjacent growth lines of the knee, stomach mucosa, small intestine, spleen and liver was the same in both groups. In contrast, the uptake of the cells of the thymus was smaller in the adjuvant-treated rats with a probability of over 90 per cent. This suggests that the mitosis of adjuvant-induced thymic cells is smaller than in cells with a normal condition.

Arthritis Induced with Freund's Adjuvant and its Relationship to Changes in the Stomach, Small Intestine and Thymus: A Histological and Tritiated Thymidine Study on Rats

The aim of the study was to elucidate histological changes provoked by Mycobacterium butyricum toxine in the rat joints, stomach, small intestine and thymus and the uptake of tritiated thymidine. Freund's complete adjuvant was injected into the base of the vastus muscle of 35 rats; 21 rats served as controls. Fifteen rats displayed histologically mild synovitis in the ankle joint, two periostitis and three muscle infiltrates. Histological study revealed no difference between the test animals and the controls in the stomach, small intestine and thymus. The thymidine uptake of the adjacent growth lines of the knee, stomach mucosa, small intestine, spleen and liver was the same in both groups. In contrast, the uptake of the cells of the thymus was smaller in the adjuvant-treated rats with a probability of over 90 per cent. This suggests that the mitosis of adjuvant-induced thymic cells is smaller than in cells with a normal condition.

The frequency of mutants in human fibroblasts UV-irradiated at various times during S-phase suggests that genes for thioguanine- and diphtheria toxin-resistance are replicated early

Human cells deficient in rate of excision repair of DNA damage induced by UV-radiation, i.e., xeroderma pigmentosum (XP) cells, are much more sensitive to the mutagenic effect of UV than are cells from normal persons. The lower frequency of mutants in the latter cells has been attributed to the fact that, unlike XP cells, they excise most of the potentially mutagenic lesions before these can be converted into mutations. If semi-conservative DNA synthesis on a template still containing unexcised lesions is responsible for introducing mutations and if replication of the gene of interest, e.g., hypoxanthine (guanine)phosphoribosyltransferase (HPRT) for thioguanine resistance or the elongation factor 2 (EF-2) for diphtheria toxin resistance, occurs at a particular time during S-phase, it should be possible to shorten the time available for such repair by synchronizing cells and irradiating them just as the gene is to be replicated. The predicted result would be a much higher frequency of mutants at one part in the S-phase than at other times. To test this, cells were synchronized using the α-polymerase inhibitor aphidicolin, which blocks cells at the G 1/S border. Autoradiography, cytoflurimetry, and incorporation of tritiated thymidine studies showed that DNA synthesis started immediately after release from aphidicolin and was completed in 8–10 h. Cells irradiated with 6 J/m 2 at various times post-release were assayed for survival and mutations. The frequency of thioguanine- or diphtheria toxin-resistant cells in the population was highest in cells irradiated during thrst fifth of the S-phase, i.e., 0–1.5 h post-release. It was significantly lower in cells irradiated at later times. In contrast, UV-induced cytotoxicity showed no significant time dependence during S-phase. These data suggest that the HPRT and EF-2 genes are replicated early in S-phase.

Histogenesis of the superior colliculus of the albino rat: A tritiated thymidine study

The pattern of generation of neurons in the albino rat superior colliculus has been studied in adult and fetal material. Neurons are generated between embryonic days 12 to 17, with rostrolateral colliculus in advance of caudomedial parts. More of the cells contributing to the deeper layers are generated early, while more of the later generated cells are located superficially. The cells of individual laminae are not formed on specific days as in the cortex, nor are the complicated gradients described previously for the chick optic tectum 18 evident. While the largest cells (found deep in the colliculus) are among the first formed and the small marginal cells among the last, there is in general a broad range of cell size being generated at any one time. The observed patterns are consistent with the concept of simultaneous production of several cell types from the ventricular epithelium on any given day. Studies of material at short times after [ 3H]thymidine injection allow correlation of the time of arrival of cells in their appropriate layer with time of arrival of afferents. In addition they suggest that factors controlling the final placement of cells in the mature nervous system is a very complex process and may involve some form of intercellular recognition.

A cytokinetic model for heterogeneous mammalian cell populations. III. Tritiated thymidine studies: Correlations among multiple kinetic parameters in human tumors

A computer model for the simulation of radioautographic studies in growing mammalian cell populations was used to study multiple tritiated thymidine ( 3HTdR) associated parameters in model populations whose properties are comparable to those found in human tumors. Several different DNA synthesis rate patterns are distinguishable, designated type I, intermediate, and type II. Correlations among percent labelled mitosis (PLM) curves, interphase cell labelling patterns, and continuous 3HTdR labelling studies suggest a type I pattern in human breast cancer, an intermediate pattern in human melanoma, and a type II pattern in human adult leukemia. Detailed simulation studies were carried out in human adult acute leukemia and human melanoma. It was possible to fit all available kinetic data in leukemia and melanoma, both with respect to low threshold data, and with respect to radioautographic labelling intensity, provided that simulated experimental conditions and simulated radioautographic conditions corresponded to those actually employed. Kinetic differences between leukemia and melanoma were demonstrated which are in keeping with the natural histories and clinical drug response behavior of these two malignancies.

Effects of theophylline and dibutyryl cyclic AMP on proliferation and keratinization of human keratinocytes

Recent evidence suggested that cyclic adenosine 3',5'-monophosphate (cAMP) may be involved in the regulation of cell proliferation and differentiation. The effects of theophylline and dibutyryl cAMP on cell division and keratinization of human epidermal cultures were examined. Nine to 12-day-old cultures were treated with these agents, separately and in combination, for various intervals. Both agents, either singly or in combination, depressed mitoses. The maximum mitotic inhibition was obtained in cultures treated with theophylline or theophylline plus dibutyryl cAMP. Tritiated thymidine studies showed that the test agents had no effect on labelling index (LI) at 4 h, but a 74% inhibition of LI was observed at 24 h. The maximum inhibition of LI (93%) occurred at 96 hours. In contrast to the control cultures, which rarely contain keratohyaline granules (KG), a marked increase in the production of these granules occurred in cultures treated with dibutyryl cAMP plus theophylline. The KG were present over the whole outgrowth. Theophylline alone also stimulated the production of KG, whereas dibutyryl cAMP had no effect. These data show that these agents inhibit cell division and this inhibition may be accompanied by an increased production of KG.

A cytokinetic model for heterogeneous mammalian cell populations: II. Tritiated thymidine studies: The per cent labeled mitosis (PLM) curve

A model is described for the simulation of tritiated thymidine radiotracer studies in mammalian cell populations, which takes into account intracycle age-specific variations in the rate of DNA synthesis and tritiated thymidine incorporation, and their relation to tritium detection characteristics by radioautographic methods. The per cent labeled mitosis curve is considered in detail. The model suggests that the per cent labeled mitosis curve alone does not contain sufficient information for the precise characterization of age structure and growth behavior in heterogeneous cell populations; however, in many instances certain features can be identified which permit the classification of population growth behavior in accordance with broad, but nonetheless useful criteria.

High dosage pre-operative radiation and surgery for carcinoma of the larynx and laryngopharynx. A 14-year program.

AbstractA carefully planned clinical program of combined pre‐operative radiation and surgery has been conducted by the Department of Otolaryngology at The Mount Sinai Hospital for the past 14 years in an effort to improve the survival rates for advanced cancer of the larynx and laryngopharynx.The Combined Therapy Program, introduced in 1958, encompassed three separate but interdependent phases. The first stage consisted of a strict protocol of 5,500 rads of Cobalt 60 teletherapy administered over a five to six‐week period. The second stage involved a rest period of three to six weeks to allow for proper healing of radiation reactions. The third stage comprised the radical procedure which included a wide field laryn‐gectomy, ipsilateral hemithyroidectomy and radical neck dissection. Whenever indicated, a contralateral neck dissection was performed as soon as feasible.The protocol described above was applied only to those patients who fit all the criteria that categorized them as having advanced cancer of the larynx and laryngopharynx. In this study all patients had a biopsy proven diagnosis of squamous cell carcinoma.A very careful statistical analysis has been made of the survival experience of this series of cases. The three and five‐year survival rates have been computed by the actuarial method,1 which when compared to the direct method, adds more reliability to the results. The direct method removes those cases not treated at least three and five years ago in computing three and five‐year survival rates, while the actuarial procedure includes all cases in the computation of survival rates.Between November, 1958, and March 1, 1972, 64 patients had been treated by combined therapy. According to the American Classification2 the 64 cases are composed of 20 Stage II (T2N0 excluded), 12 Stage III and 32 Stage IV cases. According to the actuarial method the absolute survival rates for all cases were 77 percent and 59 percent for three and five years respectively. The corresponding determinate rates were 88 percent and 86 percent. Analyzing the results according to stages, the three‐year determinate rates ranged from 100 percent for Stage II to 79 percent for Stage IV. The corresponding absolute rates ranged from 89 percent to 70 percent. The five‐year determinate rates ranged from 100 percent to 74 percent, while the absolute five‐year survival rates ranged from 50 percent for Stage II to 66 percent for Stage IV. This apparent reversal is far from being statistically significant because of considerable sampling error.The complications seen in patients treated with combined therapy have been essentially the same as those patients who undergo radical surgery without pre‐operative radiation. Several preventive measures have been utilized during surgery, and it is to be emphasized that there have been no deaths related to complications, and all patients ultimately healed completely.The important controversy which exists in the‐ combined method of therapy concerns the question of using low dosage pre‐operative radiation in the order of 1,000 to 3,000 rads or high dosages of radiation. It is our contention that serial section studies of biopsied laryngeal specimens labelled with tritiated thymidine tend to disprove the claims made favoring the value of low dosage radiation. The in vitro tritiated thymidine studies demonstrated that active DNA synthesis was observed in cancer cells in an appreciable number following dosages of 3,500 and also 5,500 rads.3 A further significant factor to be considered, we believe, is the effect of high dose pre‐operative radiation on the incidence of cervical recurrence. In this series, 44 patients had clinically palpable nodes pre‐operatively. There were six patients who developed local cervical recurrence, a rate of 14 percent.In conclusion, our statistics seem to indicate that our combined method of therapy has improved the survival rates of patients with advanced cancer of the larynx and laryngopharynx.

CANCER THERAPY-Reply

Dr. Goldman and I agree that it is not necessarily true that demonstration of the incorporation of thymidine into the new DNA of cancer cells demonstrates that these cells will necessarily divide and continue replicating DNA. No study is currently capable of a teleologic description of what cells will do. We would need a crystal ball for this purpose. However, as we indicated in the articles on tritiated thymidine studies, ie, The Intranuclear Tracer, Cell Viability, and Pre-Operative Irradiation, ( Laryngoscope , vol 78, No. 8, August 1967) and Tritiated Thymidine Studies of Radiated Laryngeal Cancer, the manufacture of DNA is probably the most important function that a cell can subserve. No cell division would be possible without first accomplishing DNA synthesis and we feel that it is a reasonable criterion of current viability. As to what will happen to the cell following DNA synthesis and one generation of cell division,

Studies of the peripheral blood of the Port Jackson shark (Heterodontus portusjacksoni) with particular reference to the thrombocyte.

Summary. Morphological features of the peripheral blood of the ancestral elasmo‐branch Heterodontus portusjacksoni, a shark species, are described. The red cell is nucleated and contains many membrane‐lined vacuoles which are derived from mitochondria. The granulocytic series resembles that of the mammals in appearance and in some phagocytic properties. The thrombocytes possess a number of similarities to mammalian platelets, but exhibit many differences. They aggregate readily, but this is temperature reversible and is independent of thrombin and ADP. They show a remarkable affinity for latex particles. Unlike many other species, fibrin formation and thrombocyte aggregation are apparently independent phenomena occurring in parallel rather than in series. Tritiated thymidine studies and the observation of mitotic figures in the peripheral blood suggest that at least part of the red cell and thrombocyte proliferation occurs in the circulating blood.

Arthritis Induced with Freund's Adjuvant and its Relationship to Changes in the Stomach, Small Intestine and Thymus: A Histological and Tritiated Thymidine Study on Rats

SummaryThe aim of the study was to elucidate histological changes provoked by Mycobacterium butyricum toxine in the rat joints, stomach, small intestine and thymus and the uptake of tritiated thymidine. Freund's complete adjuvant was injected into the base of the vastus muscle of 35 rats; 21 rats served as controls. Fifteen rats displayed histologically mild synovitis in the ankle joint, two periostitis and three muscle infiltrates. Histological study revealed no difference between the test animals and the controls in the stomach, small intestine and thymus. The thymidine uptake of the adjacent growth lines of the knee, stomach mucosa, small intestine, spleen and liver was the same in both groups. In contrast, the uptake of the cells of the thymus was smaller in the adjuvant-treated rats with a probability of over 90 per cent. This suggests that the mitosis of adjuvant-induced thymic cells is smaller than in cells with a normal condition.

SOME MORPHOLOGIC AND KINETIC STUDIES OF THE DEVELOPING ERYTHROID CELLS OF THE COMMON GOLDFISH, CARASSIUS AURATUS

ABSTRACTDeveloping erythroid cells of the goldfish Carassius auratus were obtained from kidney prints and from smears of the peripheral blood. All preparations were stained with the May‐Grunwald Giemsa technique. Developing cells were divided into six different stages. The criteria used to stage the cells were degree of chromatin condensation, degree of basophilia, nuclear:cytoplasmic ratio, and cell shape. The morphology of the maturation sequence for erythroid cells in this organism was similar to that found by other workers in other non‐mammalian vertebrates.Fish received intraperitoneal injections of tritiated thymidine, tritiated uridine or tritiated leucine so that the stages involved in DNA synthesis, RNA synthesis and protein synthesis could be determined by means of autoradiography. For the tritiated thymidine studies the per cent labeled cells per stage from four different series receiving 0.5, 1.0, 3.0 or 6.0 μCi/g body weight were pooled, since subjecting the average per cent labeled cells per stage at the lowest and at the highest dosages to Student's t‐test showed no significant differences. In all four series the fish were killed 2 hr, 12 hr and daily, 1–8 days post‐injection. The 3H‐TdR studies showed that stages I‐IV were engaged in DNA synthesis; they also showed that about 5 days were required for the stage V cell to become a mature erythrocyte (stage VI cell).Tritiated uridine was injected at a dosage of 5.0 μCi/g body weight and animals were killed 1/2, 1, 3 and 6 hr post‐injection. Grain counts showed that stages I‐IV are engaged in RNA synthesis and that the rate of this synthesis decreased as maturation proceeded.Tritiated leucine was administered at a dosage of 5.0 μCi/g body weight, and fish were killed 45 min and 3 hr post‐injection. Grain counts indicate that stages I‐V are engaged in the synthesis of protein (assumed to be globin). The fact that DNA and RNA synthesis ceased with stage IV cells while protein synthesis continued into stage V cells indicated that the mechanism responsible for protein synthesis in stage V cells was produced at an earlier stage and was self‐sustained for about 5 days.

Tritiated thymidine studies of radiated laryngeal cancer.

CLINICAL evaluation of the effectiveness of radiotherapy on advanced cancer of the larynx has proven precarious. 1 We are familiar with patients in whom sizeable tumors have disappeared clinically following radiotherapy, only to reappear and necessitate attempts at salvage. Similarly, microscopic nests of apparently viable cells often appear in the larynges of patients who have received tumoricidal range dosages of radiation followed by laryngectomy and neck dissection. 2 Since the effects of radiotherapy are determined primarily by ionizing events within cells, it is logical that this treatment be followed at cellular and radiobiologic as well as clinical and pathologic levels. To this end we are currently using tritiated thymidine autoradiography to monitor the effects of preoperative radiation on the death and recovery of cancer of the larynx. 3 Cell viability can be established unequivocally by the presence of intranuclear tritium, and more important, predictions of the behavior of radiated tumor

Effects of Freezing on the Gastric Mucosa of Dogs

Summary Gastric freezing for 1 hour at -20° C. inflow and -12° C. outflow was performed in dogs with gastrostomies. There was no significant reduction in acid secretory response to Histalog, insulin, or peptone after freezing. Studies by light and electron microscopy showed a very transient and patchy damage to the superficial layers of the mucosa with no effect on the glandular layers. Tritiated thymidine studies showed normal cell renewal within 1 week after freezing. After 7 1-hour freezes, the gastric mucosa appeared histologically normal and acid secretion was present. The rationale of the method in the treatment of peptic ulcer is questioned.