DIFFERENCES IN REUTILIZATION OF THYMIDINE IN HEMOPOIETIC AND LYMPHOPOIETIC TISSUES OF THE NORMAL MOUSE

Swiss Albino mice received a single i.v. injection of ³H-thymidine (TdR) or of ¹²⁵I-deoxyuridine (IUdR). Bone marrow, thymus, spleen and mesenteric lymph node were examined for the efficiency of precursor incorporation into DNA, and for DNA renewal from day 1 to day 8.
TdR is 5–8 times more efficiently incorporated by the different organs in vivo and in vitro than is IUdR. This indicates that the discrimination against IUdR occurs at the level of DNA synthesizing cells.
A diminished DNA turnover rate measured with ³H-TdR in comparison with ¹²⁵I-UdR is interpreted to indicate reutilization of TdR.
TdR reutilization was observed in bone marrow and spleen from at least day 1 on, and in the thymus from day 3 on, following pulse labeling of DNA synthesizing cells. The degree of TdR reutilization appears higher in the thymus (67%) than the bone marrow (43%) and spleen (38%). The mesenteric lymph node indicates either no, or a very low efficiency of TdR reutilization. The data are also consistent with a reutilization equally efficient for TdR and IUdR.
It is suggested that the TdR salvage pathway in hemopoietic tissues is largely localized to single organs which have immediate access to TdR made available by catabolism of DNA. The contribution of TdR from systemic reutilization to the organs studied falls within the limits of error of measurements. Moreover, the TdR salvage pathway especially in the lymph node may involve other DNA breakdown products than nucleosides.     

Study of cell death in Friend leukaemia.

Cell death of splenic Friend leukaemic cells has been studied in vivo, using 125I-UdR and 3H-TdR pulse labelling. The evolution of the splenic specific activity has been measured by autoradiography and external counting during 40 hr after injection of the labelled precursor. These two techniques show the existence of a large reutilization of 3H-TdR (50%), which is measurable as soon as 7 hr after the injection. The DNA turnover rate is rapid, 83-8% of the splenic cellular DNA being renewed per day. Those results confirm that most of the cells produced in the Friend leukaemic spleen are rapidly lost; they also demonstrate that this cell loss is mainly due to a massive death, which occurs in proerythroblastic and erythroblastic compartments after one or two cell divisions. Friend leukaemic cells, which are characterized by a limited capacity of proliferation and a short lifespan, do not appear to be malignant.     

KINETICS OF NEUTROPHILIC GRANULOCYTES IN THE BLOOD OF RATS

The kinetics of neutrophilic granulocytes in the blood of rats were investigated using in vivo ³H-TdR labelling and autoradiography. The radioactive precursor was administered by single injection, repeated injections at 5 hr intervals and continuous infusion. The appearance of labelled granulocytes in the blood and in the sputum was recorded up to 120 hr after tracer application. In contrast to results after a single injection of ³H-TdR, complete labelling of the blood granulocyte pool was achieved when the DNA precursor was given by continuous infusion or four repeated injections at 5 hr intervals. In the latter experiments, an exponential replacement of unlabelled blood granulocytes by labelled granulocytes could be demonstrated, the mean intravascular half-life being 5·7 hr. This figure is in good agreement with values obtained by isotopic techniques in other mammalian species.     

STUDY OF CELL DEATH IN FRIEND LEUKAEMIA

Cell death of splenic Friend leukaemic cells has been studied in vivo, using ¹²⁵I-UdR and ³H-TdR pulse labelling. The evolution of the splenic specific activity has been measured by autoradiography and external counting during 40 hr after injection of the labelled precursor. These two techniques show the existence of a large reutilization of ³H-TdR (50%), which is measurable as soon as 7 hr after the injection. The DNA turnover rate is rapid, 83-8 % of the splenic cellular DNA being renewed per day. These results confirm that most of the cells produced in the Friend leukaemic spleen are rapidly lost; they also demonstrate that this cell loss is mainly due to a massive death, which occurs in proerythroblastic and erythroblastic compartments after one or two cell divisions. Friend leukaemic cells, which are characterized by a limited capacity of proliferation and a short lifespan, do not appear to be malignant.     

Cellular proliferation kinetics of the murine sarcoma induced by the Moloney sarcoma virus

Abstract. Cell proliferation kinetics of the sarcoma induced by Moloney virus was studied in newborn Swiss OFl mice.
After in vivo injection of tritiated thymidine, followed by autoradiography, it was shown that the majority of cells were actively proliferating (labelling index; 31%, growth fraction 78%). The mean cell cycle was 16 hr and cell loss was relatively low (cell loss factor 48%). The study of tumour specific activity with time after a single [ in vivo ] injection of [³H]dR or [¹²⁵I]UdR did not demonstrate the same degree of cell loss as that calculated by autoradiography. This result is consistent with a massive reutilization of radioactivity released by normal tissues.     

DIFFERENT ACTION OF SUICIDAL DOSES OF TRITIATED THYMIDINE AND HYDROXYUREA ON MURINE HAEMOPOIETIC CELLS

In order to gather information on the factors that cause the different action of suicidal doses of tritiated thymidine (³H-TdR) and of hydroxyurea on murine stem cells, the incorporation of ³H-TdR into DNA of bone marrow and spleen cells has been studied. Continuous death of labelled cells after suicidal ³H-TdR is indicated by a more pronounced decline of total DNA-bound radioactivity in bone marrow and spleen cells compared to that in control animals which had received tracer doses of ³H-TdR. Extensive and rapid loss of DNA-bound radioactivity occurred in ³H-TdR labelled animals after hydroxyurea treatment indicating an instantaneous and highly effective killing of labelled cells. After double labelling of DNA with ³H-TdR and ¹²⁵iodo-deoxyuridine (¹²⁵I-UdR), the decline of the ratio of DNA-bound ¹²⁵I to DNA-bound ³H after suicidal ³H-TdR indicates prolonged tritium reutilization. Following hydroxyurea, reutilization was completed within the first 12 hr after drug administration. These findings explain in part the slow recovery of different stem cell compartments after suicidal ³H-TdR on the basis of protracted tritium reutilization as compared to the fast recovery which follows the rapid action of hydroxyurea.     

CELL LOSS FROM SEVERAL TYPES OF SOLID MURTNE TUMOUR: COMPARISON OF 125I-IODODEOXYURIDINE AND TRITIATED THYMIDINE METHODS

The method of measuring tumour cell loss rates in situ following radioactivity loss after a single injection of ¹²⁵I-iododeoxyurudine (¹²⁵I-UdR) was tested for its accuracy in five different types of murine tumour. To achieve this the method was compared with two others: (1) using ¹²⁵I-UdR, but excising tumours before the radioactivity determinations, with or without extracting DNA; (2) using tritiated thymidine and autoradiography. A third method was used on three of the tumours, in which ¹²⁵I-UdR-labelled tumours were grown in unlabelled hosts, followed by whole body counting of the tumour-bearing mice. In two of the tumours an increase was observed in total tumour radioactivity with time after ¹²⁵I-UdR injection. This prevented the estimation of cell loss parameters in these tumours. Approximately half the increase was due to reutilization of ¹²⁵I-UdR supplied from tissues within the mouse; approximately a third to an influx of labelled inflammatory cells (probably in response to infection accompanying ulceration of overlying skin); and the remainder to an increase in non-DNA radioactivity. In these tumours cell loss rates could be obtained from the whole body counting technique in which influxes of labelled cells and reutilizable radioactivity were eliminated. A comparison of either ¹²⁵I-UdR technique with the ³H-TdR technique showed good agreement of the cell loss factors for the low cell loss tumours. However, for tumours with high cell loss factors the ¹²⁵I-UdR technique gave lower values for cell loss. This implied that reutilization of ¹²⁵I-UdR within the tumour (i.e. from internal, not external sources) occurred in the high cell loss tumours. It is concluded that equating radioactivity loss with cell loss after an injection of ¹²⁵I-UdR is reasonable for some tumours, but will result in significant underestimates in others. For high cell loss tumours the ³H-TdR technique will give the     

COMPARISON OF DNA RENEWAL IN GERM-FREE AND CONVENTIONAL MICE USING [125I]IODODEOXYURIDINE AND [3H]THYMIDINE

Germ-free (GF) and conventional (CV) C3H mice received a single injection of 1 μCi [³H]thymidine and 3 μCi [¹²⁵I]iododeoxyuridine to provide simultaneous labeling of DNA with the two precursors. Thymus, spleen, mesenteric lymph nodes, bone marrow (femora), small intestine, colon and skin were examined for total organ activity and rate of DNA renewal 1–8 days after injection. Precursor incorporation, assayed on day 1, was lower in the thymus, mesenteric lymph nodes and femora (and, to a lesser extent, in the spleen and colon) of GF mice as compared to CV animals. The opposite was observed in the small intestine and skin, i.e. total organ activity was higher in GF animals. Differences in precursor incorporation were partly due to differences in organ weights between the two groups of mice. In comparison to CV animals, DNA renewal rates were diminished in the mesenteric lymph nodes, bone marrow, colon (following a 3-day plateau) and spleen of GF mice. Little, if any, difference was observed between the two groups with respect to the rate of DNA turnover in the thymus and skin. Radioactivity of the small intestine remained constant for 2 days. Thereafter intestinal activity in GF mice declined at an initial slow rate between days 2 and 5 followed by a rapid decrease between days 5 and 8. In CV mice the first phase of activity loss was short with the rapid decline in intestinal activity beginning on day 3. From the slopes of the regression lines, the percentage thymidine reutilization was estimated. Reutilization varied from 0 to 63% in the various organs examined, with the greatest difference between GF and CV mice occurring in the mesenteric lymph nodes.     

CELL LOSS AND INFLUX OF LABELED HOST CELLS IN THREE TRANSPLANTABLE MOUSE TUMORS USING [125I]UdR RELEASE

The [¹²⁵I]UdR loss technique was used to estimate cell loss from RIF-1, EMT6 and KHJJ tumors in order to determine the length of the delay between labeling and the beginning of the loss of labeled cells, and also to calculate a value for ø, the cell loss factor. To determine the importance of reutilization of label released from the gut and/or the influx of labeled host cells, the blood flow to some tumors was occluded during and for 30 min after injection of the label. Relatively small amounts of radioactivity entered occluded RIF-1 tumors during 9 days after injection of [¹²⁵I]UdR, indicating that reutilization of systemic label and influx of labeled host cells are not significant in this system. In contrast, substantial amounts of radioactivity entered occluded EMT6 and KHJJ tumors, reaching 40% of the total activity in non-occluded tumors during 6 days following injection. After corrections were made for this influx of label, the [¹²⁵I]UdR loss curves from RIF-1 and EMT6 tumors were essentially exponential from the first day following injection of label. This was interpreted as indicating the loss of proliferating as well as non-proliferating cells from both tumors. The cell loss factor derived from the [¹²⁵I]UdR loss curves corrected for influx appeared to agree well with published values derived from analysis of percent labeled mitoses curves. In contrast, the corrected [¹²⁵I]UdR loss curves from KHJJ tumors showed that loss of activity began three days after injection of label, indicating that primarily nonproliferating cells are lost from this tumor.     

The dynamic state of liver gap junctions

By the use of a simple, rapid method for the isolation of gap junctions from small amounts of rat liver (2–3 g), we have followed the incorporation of the radiolabeled amino acid precursors ³H-leucine and ³⁵S-methionine into the gap junction protein. In timed studies with ³⁵S-methionine as precursor, the specific activity in the protein is maximal by 4 h after a single injection of 300 μCi/100 g body weight. From the decay in the specific activity with time after a single injection, the gap junction protein has an apparent half-life of about 19 h. Because of problems of reutilization of radiolabeled amino acid with ³⁵S-methionine as precursor, this apparent halflife probably overestimates the true half-life and indicates a surprisingly rapid turnover of the gap junction protein. This short half-life suggests that, in rat liver, the gap junctions may be very responsive to alterations in physiological demands.     

Reprogramming in the absence of DNA synthesis in Galleria larval epidermis.

Larval epidermal cells from a day-1 penultimate instar Galleria larva on implantation into day-5 last instar larva metamorphose and deposit a pupal cuticle at the same time as the host pupates. DNA synthesis in the implanted larval cell was monitored with 3-H-thymidine. Various regimens of 3-H-thymidine application were used and under no conditions did the larval cells incorporate label during the period from implantation to deposition of pupal cuticle. This suggests that a wax moth larval ectoderm cell can reprogram its genome to secrete a pupal cuticle without a precedent cell division.     

Uptake of 125I-iododeoxyuridine in mouse organs during deuteration of body water: Evidence for a thymus-specific effect

The effects of body water deuteration on mammalian DNA synthesis $\text{in vivo}$ during the deuterium equilibration period in the body were studied. Young adult mice were given 15% or 30% D₂O in the drinking water for 4, 10 or 21 days. Control mice were given distilled water. Eighteen hours prior to sacrifice, ¹²⁵IUdR, a conveniently monitored synthetic analogue of the DNA precursor thymidine, was injected intravenously. Although neither radioiodine activity of the total body nor body weight varied significantly among the three groups, thymic radioactivity per g tissue was significantly lower in mice given 30% D₂O and, to a lesser extent, in mice given 15% D₂O than in the control group. In contrast, intestine and hemopoietic bone marrow displayed minor changes in ¹²⁵IUdR incorporation. This reduction of ¹²⁵IUdR incorporation is discussed in relation to the particular importance of thymidine reutilization in the thymus.     

Assessment of DNA damage in canine peripheral blood and bone marrow after total body irradiation using the single-cell gel electrophoresis technique

DNA damage in single peripheral blood (pb) and bone marrow (bm) cells was studied in dogs which were exposed to total body X-ray irradiation (TBI) with a lethal dose of 3.9 Gy. The changes in pb and bm cell numbers were measured within 9 days after TBI. Using the alkaline single-cell gel electrophoresis technique (‘comet’ assay). DNA strand breaks and alkali labile sites were assessed in single cells derived from the blood before TBI, 1 h and 4 h after TBI and on days 1, 3 and 9 after TBI. Bone marrow cells subjected to the assay were collected before and on days 1 and 9 after TBI. Cells expressing the strongest DNA damage were most frequent in the blood 1 h after TBI and in the bone marrow 1 day after exposure. Thereafter, a continuous reduction of DNA damage in individual cells was observed in the course of progressive leukopenia and granulocytopenia.     

Ribonucleotide reductase--a "target" of the action of nitrosomethylurea]

The antitumor and toxic effects of methylnitrosourea (MNU) are determined through its metabolic pathways. In organism MNU is subject to hydrolytic decomposition and denitrosation. It has been shown in vivo studies that MNU abdominal injections of therapeutic doses caused the inhibition of ribonucleotide reductase in mouse spleen, and therefore the DNA synthesis depress. The effect may apparently contribute to antitumor property of MNU. It has been estimated that destruction of M2 subunit of the enzyme is occurred. The relation between the loss of ribonucleotide reductase activity and the inhibition of protein synthesis was discussed. Besides, the cancerogenic and mutagenic properties of MNU were discussed as a result of imbalance of DNA precursor pools. Changes in contents of Fe(3+)-transferrin, ceruloplasmin, methemoglobin in blood and spleen of animals after MNU injections have been found. The changes were reversible after single MNU injection and became irreversible after multiple injections.     

The Effect of X-Irradiation On Cell Loss In Five Solid Murine Tumours, As Determined By the 125Iudr Method

The rate of cell loss in irradiated RIF-1, EMT6, KHJJ, B16 and KHT tumours was studied using the ¹²⁵IUdR loss technique. Administration of ¹²⁵IUdR preceded localized tumour irradiation by 2 days. Loss of tumour radioactivity was measured for 6–8 days after irradiation. the blood flow to some tumours was occluded during, and for 30 min following, injection of the label to measure the amount of radioactivity entering the tumour as a result of reutilization of label from the gut epithelia and influx of labelled host cells. Irradiation did not significantly alter the amount of radioactivity entering these clamped tumours during the 8–10 days after injection of ¹²⁵IUdR. This permitted comparison of irradiated and control groups based on the loss of radioactivity from the non-occluded tumours. Irradiation of RIF-1, EMT6, KHJJ or B16 tumours with doses of 600, 1400, 2400 or 4400 rads produced no significant increase in the rate of loss of tumour radioactivity. This suggested that, in the population of labelled cells, cell lysis following irradiation proceeded slowly. In contrast, KHT tumours showed a significant increase in loss rate following each radiation dose, although the increase was dose-independent. In all tumour systems, the constant rate of cell loss after radiation appeared to coincide with published reports of tumour growth responses after irradiation. the present data suggest that the manner of expression of radiation-induced cell killing results from the cellular proliferative status, i.e. whether a cell is cycling or non-cycling.     

Incorporation of thymidine and iododeoxyuridine into the DNA of mouse tissues.

Mice were injected intravenously with a solution containing tritiated thymidine (TdR) and iodine-labelled iododeoxyuridine (IUdR). The ratio of 3H/125I activities was measured in the acid-soluble fraction and in the DNA of several tissues at various times from 0.08 to 24 h after injection. There did not appear to be any discrimination in favor of TdR in the acid-soluble fraction of the tissues. The amount of TdR incorporated into the DNA was four to five times greater than the amount of IUdR incorporated; moreover, this value remained relatively constant throughout the period of DNA synthesis under the conditions used. Although IUdR was destroyed more rapidly than TdR in the body, particularly at high concentrations of both precursors, this factor did not account for the major portion of the discrimination observed with tracer amounts of the two DNA precursors. Discrimination in favor of TdR as a precursor for DNA must, therefore, occur at some stage in the utilization of intracellular precursor.     

FURTHER OBSERVATIONS ON THE LATE LABELLING ASSOCIATED WITH STIMULUS-RESPONSIVE CELLS IN SKIN

The presence of a long-lived DNA precursor pool which may show some specificity for stimulus-responsive cells has been demonstrated. Autoradiography, biochemical analysis, hydroxyurea sensitivity, and the temporal response all suggest that the late incorporation is into DNA in cells in the basal layer of the skin that respond to stimulation. The effect is observed with various doses of tritiated thymidine and both methyl and 6 labelled material. ¹²⁵Iododeoxyuridine also shows late labelling in skin. It is believed that the late labelling is readily distinguishable from reutilization and from possible slow utilization of catabolites into molecules other than DNA. Skin of the ear and dorsum shows similar degrees of late incorporation, while tail and foot skin apparently have smaller long-lived pools. This may indicate smaller stimulus-responsive cell populations. If tritiated thymidine is given to cells after stimulation there is still some slow or delayed uptake. The distribution of labelled cells in the autoradiographs suggests that the late labelling cells may be associated somehow with pre-existing labelled cells (cells in S at the time of thymidine administration).     

Cell kinetics methods for clinical use: assessment of autoradiography and 125I-iodo-deoxyuridine with mouse Ehrlich tumour.

Superficial Ehrlich tumours in mice were used to assess how much information on cell kinetics could be obtained from only the simplest techniques of autoradiography, and in situ monitoring of 125I-iodo-deozyuridine. These techniques were selected as being readily applicable to clinical situations. Intradermal tumours were studied from the earliest stages of rapid growth to large slow growing tumours with necrotic cores, as well as tumours undergoing regression. For comparison, intramuscular tumours were studied with systemic injections of radioactive DNA precursors. It was found that extensive information on cell production and loss rates was obtained from a single injection of tritiated thymidine followed by a single biopsy, or alternatively in vitro labelling of one minute biopsy specimen, and measurements of only the labelling index, together with a knowledge of the tumour's growth characteristics. Following a single localized injection of 125I-iodo-deoxyuridine, the loss rate of radioactivity measured in situ for a period of about 1 week provided an index of cell loss rate from the tumours.     

Transcellular transport of polymeric IgA in the rat hepatocyte: biochemical and morphological characterization of the transport pathway

Polymeric IgA (pIgA) is transported by liver parenchymal cells (hepatocytes) from blood to bile via a receptor-mediated process. We have studied the intracellular pathway taken by a TEPC15 mouse myeloma pIgA. When from 1 microgram to 1 mg 125I-pIgA was injected into the saphenous vein of a rat, 36% was transported as intact protein into the bile over a 3-h period. The concentration of transported 125I-pIgA was maximal in bile 30-60 min after injection, and approximately 80% of the total 125I-pIgA ultimately transported had been secreted into bile by 90 min. A horseradish peroxidase-pIgA conjugate (125I-pIgA-HRP) was transported to a similar extent and with kinetics similar to that of unconjugated 125I-pIgA and was therefore used to visualize the transport pathway. Peroxidase cytochemistry of livers fixed in situ 2.5 to 10 min after 125I-pIgA-HRP injection demonstrated a progressive redistribution of labeled structures from the sinusoidal area to intermediate and bile canalicular regions of the hepatocyte cytoplasm. Although conjugate-containing structures began accumulating in the bile canalicular region at these early times, no conjugate was present in bile until 20 min. From 7.5 to 45 min after injection approximately 30% of the labeled structures were in regions that contained Golgi complexes and lysosomes; however, we found no evidence that either organelle contained 125I-pIgA-HRP. At least 85% of all positive structures in the hepatocyte were vesicles of 110-160-nm median diameters, with the remaining structures accounted for by tubules and multivesicular bodies. Vesicles in the bile canalicular region tended to be larger than those in the sinusoidal region. Serial sectioning showed that the 125I-pIgA-HRP-containing structures were relatively simple (predominantly vesicular) and that extensive interconnections did not exist between structures in the sinusoidal and bile canalicular regions.