Cytochemical localization of alkaline phosphatase in intestinal metaplasia of the human stomach

Summary Alkaline phosphatase in the brush border of areas of intestinal metaplasia of human stomach was studied cytochemically. All absorptive cells in the upper part of the villi of the duodenum had strong alkaline phosphatase activity but, in areas of intestinal metaplasia, the metaplastic glands consisted of alkaline phosphatase-positive and negative absorptive cells. Alkaline phosphatase activity was found in tall dense microvilli of absorptive cells in areas of intestinal metaplasia and in the duodenum. However, in some areas of metaplastic epithelium, the activity was very weak in some tall dense microvilli of absorptive cells but strong in those of neighbouring absorptive cells. No alkaline phosphatase activity was found in short sparse microvilli of absorptive cells in areas of intestinal metaplasia. The difference in alkaline phosphatase activity in microvilli of different cells in areas of intestinal metaplasia, which is not seen in the duodenum, indicates abnormal morphological and enzymatic differentiation in intestinal metaplasia.     

Studies on lung tumours. IV. Correlation between [3H]thymidine labelling of lung and liver cells and tumour formation in GRS/A and C3Hf/A male mice following administration of dimethylnitrosamine.

Male mice of the inbred strain GRS/A are highly susceptible to lung tumour but refractory to liver tumour formation, whereas the opposite relation holds for C3Hf/A male mice. Liver and lung cells of these 2 mouse strains were studied autoradiographically after intraperitoneal injection of [3H]dimethylnitrosamine (DMN) and of [3H]thymidine at days 1--14 after administration of unlabelled DMN. Corresponding cell types in the lungs or livers of these 2 mouse strains bound similar amount of [3H]DMN. Among the various types of lung cells only the alveolar Type II cells, from which the lung adenomas derive, showed a strain-specific difference in [3H]thymidine labelling indices, much more cells becoming labelled in the case of the GRS/A than of the C3Hf/A strain at days 3--7 after carcinogen administration. Opposite thymidine labelling indices were exhibited by the parenchymal liver cells of the 2 strains, with C3Hf/A now showing a greater response than did GRS/A males. Thus thymidine-labelling and tumourigenic responses of target lung and liver cells to carcinogen in the 2 strains coincided. Sulphur dioxide and carbon tetrachloride mimicked the effects of DMN on the thymidine labelling indices of, respectively, the lung alveolar Type II and the thymidine labelling indices of, respectively, the lung alveolar Type II and the liver parenchymal cells of the 2 strains. The nature of the differential effect of carcinogen on the [3H]thymidine labelling of the cells and the correlation of these patterns with susceptibility to tumour formation, are briefly discussed.     

Differentiation of cartilage cells studied by quantitative [3H]thymidine autoradiography in vitro

The apical segments of the mandibular condylar cartilage of newborn ICR mice, containing the intact zones of progenitor cells along with a few rows of chondroblasts were initially prelabelled in vitro with [3H]thymidine and were subsequently chased and cultured for as long as eight days. Such explants underwent a process of tissue regeneration, as after three days in culture they reconstituted the original structure of the organ, thus resembling the in vivo appearance of neonatal mandibular condylar cartilage. Cellular proliferation with subsequent differentiation in the regenerating tissue was followed by means of quantitative autoradiography. Immediately after labelling, the autoradiography-positive grains were confined exclusively to progenitor cells. The latter revealed a substantial ability to proliferate in vitro, a fact that was manifested by a progressive increase in the labelling index along the course of the culture. The latter process was followed by cellular differentiation thereby obtaining hypertrophic chondrocytes. The increase in the rate of labelling index and in the total number of [3H]thymidine-labelled cells was significantly correlated with the overall growth of the regenerating explants.     

Simultaneous autoradiographic and histochemical analysis of bone formed in vitro

The simultaneous histochemical demonstration of alkaline phosphatase activity and autoradiographic demonstration of [3H]-thymidine uptake is valuable for study of bone cell kinetics in vivo or in vitro. By use of this technique, it has been possible to detect changes induced by a single dose of dexamethasone (10(-7) M) in the time course of alkaline phosphatase activity, the number of alkaline phosphatase-positive cells, and [3H]-thymidine labeling in bone formed in vitro.     

Immunofluorescent and autoradiographic analysis of the relationship between the presence of histone H5 and DNA synthesis in developing chick erythroid cells

Simultaneous detection of histone H5 by indirect immunofluorescence and of [³H]thymidine incorporation by autoradiography on the same preparations of developing erythroid cells have been used to precisely define the extent of correlation between the loss of nuclear activity and the presence of histone H5. It was found that from day 3–12 of embryonic life there are two successive waves of double-labelled cells. At some stages, as many as 30% of the cells which incorporate [³H]thymidine also contain histone H5. Thus, the simple presence of H5 cannot be sufficient to cause nuclear inactivation. A kinetic analysis of the appearance and disappearance of [³H]thymidine-labelled cells, containing histone H5, and cells which are positive for both markers is presented. The result is consistent with the interpretation that the appearance of H5 in the first wave of double labelled cells occurs just before the erythroid cells become metabolically inactive. These observations modify the concept that histone H5 functions uniquely or solely as a template repressor.     

Temporal changes in the response of chick limb bud mesodermal cells to transforming growth factor beta-type 1

Transforming growth factor beta (TGF beta) has been found in adult and developing bone in vivo and has varied effects on chondrocytes and osteoblasts in culture. We investigated the effects of TGF beta-type 1 on embryonic chick endochondral bone precursors in culture. Stage 24 chick limb bud mesoderm cells were cultured at high density. Under these conditions a dense mat containing nodules of cartilage surrounded by alkaline phosphatase-positive cells formed. Exposure of mesodermal cells to TGF beta-type 1 over the course of 4-7 days or during Days 3 and 4 caused a reduction in alkaline phosphatase activity and [35S]sulfate incorporation into proteoglycans. When the TGF beta-type 1 was applied during Days 1-2, it caused an increase in both parameters: these increases were observed in the absence of a corresponding change in [3H]thymidine incorporation. The specificity of the effects of TGF beta-type 1 was confirmed by neutralizing antibodies. These studies show that TGF beta-type 1 modulates the phenotype of embryonic endochondral bone precursors. The influence may depend on the window of exposure to the growth factor and, therefore, on the state of differentiation of the cells.     

Utilization of the label from bacterial [3H] DNA by isolated barley roots and embryos under different conditions

[³H] DNA fromEscherichia coli and [³H] thymidine were applied, in sterile conditions, on isolated barley embryos and on roots excised from these embryos, both cultivated in the liquid medium and on halves of barley seeds, through the endosperm bridge. In embryos and roots, the labelled compounds were applied in 1.5% sucrose + 0.2 SSC alone, or together with either unlabelled thymidine or DEAE-dextran. Similar labelling indices were found after [³H] thymidine and [³H] DNA treatment which shows that the activity of [³H] DNA is utilized during the S phase. After application of [³H] thymidine, only cell nuclei in S phase were labelled. After the application of [³H] DNA an extranuclear label, in addition to the labelling of nuclei in the S phase, was observed in some experimental variants. The density of label above labelled nuclei after [³H] DNA treatment sharply decreased when unlabelled thymidine or DEAE-dextran was added, while the density of label above nuclei labelled by [³H] thymidine decreased when unlabelled thymidine but not DEAE-dextran was added. The labelling of nuclei with the label from [³H] DNA is the result of degradation of exogenous DNA reutilization of low molecular weight products. Extranuclear labelling is most probably due to the polymerous or partly degraded DNA.     

RNA synthesis by villus and crypt cell nuclei of rat intestinal mucosa

Rat intestinal mucosa was separated by eversion and vibration to provide a sequence of fractions from predominantly villus cells to predominantly crypt cells. The proportions of these cell types in each fraction were computed from the concentrations of alkaline phosphatase (villus cells) and thymidine kinase (crypt cells) in each population. The isolated mucosal fractions varied from about 90% villus cells to 90% crypt cells. Following injection of the rats with [³H]thymidine, the nuclei were isolated from each mucosal cell fraction and the amount of radioactivity incorporated into DNA was measured as an index of crypt cell abundance. The isolated nuclei were also incubated with ribonucleoside triphosphates and the amount of RNA synthesized was measured. Nuclei labeled with [³H]thymidine were found only in fractions rich in crypt cells, whereas capacity for RNA synthesis remained very active in mucosal fractions consisting predominantly of villus cells. It is concluded that non-dividing villus cells continue to make RNA.     

Inhibition of cell division by interferons: Changes in the transport and intracellular metabolism of thymidine in human lymphoblastoid (Daudi) cells

The Daudi line of human lymphoblastoid cells shows a high sensitivity towards growth inhibition by human interferons. In cells pretreated with 70 reference units/ml of an interferon preparation for 48 h, the incorporation of exogenous [3H]thymidine into DNA is inhibited by as much as 85%. We are investigating the extent to which this effect reflects a true inhibition of the rate of DNA synthesis or whether it may be caused by changes in the metabolic utilization of exogenous thymidine by the cells. Interferon treatment results in a 30% inhibition of the rate of membrane transport and a 60% decrease in the rate of phosphorylation of [3H]thymidine in vivo. The latter effect is due to a decrease in V of thymidine kinase without any change in the value of Km for this enzyme. In addition to these changes, incorporation of [3H]uridine into DNA, which occurs as a result of the intracellular conversion of this precursor into thymidine nucleotides, is also inhibited by 75%, whereas RNA labelling by [3H]uridine is decreased by only 15% in interferon-treated cells. Thus several different metabolic events associated with thymidine nucleotide metabolism and DNA synthesis in Daudi cells are disrupted by interferon treatment.     

The possible role of pancreatic proteases in the turnover of intestinal brush border proteins.

1. Intestinal brush border enzymes have heterogeneous rates of turnover, the largest proteins having the fastest turnover. Since the membrane faces the intestinal lumen, the effects of pancreatic factors were examined in mediating this turnover. Surgical subtotal pancreatectomy was used as an experimental model to study the turnover of brush border proteins in the absence of most pancreatic secretions. 2. Subtotal (95%) pancreatectomy of rats was found to cause elevations by about 50% of total activity and specific activities of certain brush border enzymes (maltase, sucrase, lactase), but not of others (alkaline phosphatase, trehalase). Rats were judged to be functionally deficient in pancreatic proteolytic enzymes (a) by demonstration of vitamin B-12 malabsorption, which was corrected by trypsin, and (b) by the finding of only about 20% of proteolytic activity appearing in the lumen after a test meal when compared to control. 3. To measure protein turnover in vivo the method of double labelling was used, where [3H]- and [14C]valine were administered intraduodenally in sequence 10 h apart. With this technique, a high 3H/14C ratio is correlated with rapid turnover. Proteins with apparent molecular weights of about 200 000-270 000 were found to turn over more rapidly than smaller proteins. 3H/14C ranged from 4.7 to 6.2 in animals without pancreatic insufficiency. In the face of decreased pancreatic proteolysis, the 3H/14C ratio was 2.3-3.1, similar to that of proteins with a slow half life. 4. Estimates of relative synthetic rates of large brush border proteins were lower than normal in pancreatectomized animals, but were constant over the period of the labelling experiment. The high enzyme levels in the face of lower synthetic rates confirms that, at the new steady rate, degradation rates must be slower for large brush border proteins in pancreatic insufficiency. 5. In vitro, using purified brush borders, unfractionated pancreatic enzymes were found to remove sucrase, maltase and lactase, but not alkaline phosphatase and trehalase. The enzyme most potent in this respect was the pancreatic protease, elastase. Non-proteolytic enzymes (amylase, lipase, phospholipase A) were inactive in removing enzyme from the brush border. The addition of elastase to pancreatectomized animals in vivo restored the rapid turnover rate of large brush border proteins. 6. A model is thus proposed for the normal catabolism of some large intestinal brush border proteins. It is suggested that the surface of intestinal absorptive cells is being constantly remodelled, and that certain surface enzymes are in part removed from the membrane by the action of pancreatic proteases. A possible special role for elastase is suggested.     

Further studies on the size and composition of the chick embryo fibroblast cytosolic DNA complex

The chick embryo fibroblast cytosolic DNA complex shows anomalous elution behaviour on agarose gel column chromatography. The indicated molecular size varies between 5 X 10(5) dalton (higher exclusion limit gels) and 1.4 X 10(6) dalton (lower exclusion limit gels). Chromatography on lower exclusion limit gels shows the [3H]thymidine labelled (DNA) complex as a sharp peak, coincident with a peak of [3H]uridine and [3H]lysine labelling and similar pulse labelling patterns for the three precursors but with DNA labelling lagging behind RNA and protein. Both cultured and uncultured cell cytosols show an A260 peak coincident with the [3H]precursor labelling peaks.     

DNA synthesis in Langerhans' cells of mouse ear epithelium revealed by tritiated thymidine autoradiography and histochemical staining for non-specific esterase and beta-glucuronidase activity

The proportion of Langerhans' cells in DNA synthesis in normal mouse skin was assessed by combining tritiated thymidine [3H]TdR autoradiography with enzyme histochemistry. After injection of [3H]TdR, ear skin was treated in two ways. Epithelial sheet preparations were stained for the presence of non-specific esterase and cytospin preparations of epithelial cell suspensions were stained for beta-glucuronidase activity. The labelling index (+/- SE mean) for cytospins, 40 min after injecting [3H]TdR, was 1.6 +/- 0.15%, doubling to 3-4% from 7-17 days after injection. The sheet preparations showed the proportion of label attributable to paired Langerhans' cells rising from 18% at 40 min after injection, to approximately 45%, on days 1-4 after injection. These results suggest that the proliferation of Langerhans' cells in normal mouse skin might be higher than was previously thought to be the case.     

Ribonucleotides Linked to DNA of Herpes Simplex Virus Type 1

Cells of a continuous cell line derived from rabbit embryo fibroblasts were infected with herpes simplex type 1 virus (HSV-1) and maintained in the presence of either [5-(3)H]uridine or [methyl-(3)H]thymidine or (32)PO(4) (3-). Nucleocapsids were isolated from the cytoplasmic fraction, partially purified, and treated with DNase and RNase. From the pelleted nucleocapsids, DNA was extracted and purified by centrifugation in sucrose and cesium sulfate gradients. The acid-precipitable radioactivity of [5-(3)H]uridine-labeled DNA was partially susceptible to pancreatic RNase and alkaline treatment; the susceptibility to the enzyme decreased with increasing salt concentration. No drop of activity of DNA labeled with [(3)H]thymidine was observed either after RNase or alkali treatment. Base composition analysis of [5-(3)H]uridine-labeled DNA showed that the radioactivity was recovered as uracil and cytosine. In the cesium sulfate gradient, the purified [5-(3)H]uridine-labeled DNA banded at the same position as the (32)P-labeled DNA. The present data tend to suggest that ribonucleotide sequences are present in HSV DNA, that they are covalently attached to the viral DNA, and that they can form double-stranded structures.     

Reduced DNA repair during differentiation of a myogenic cell line

Repair synthesis induced by 4-nitroquinoline-1-oxide (4NQO) in L6 myoblasts before and after cellular fusion was measured by [3H] thymidine incorporation into unreplicated DNA. The level of repair synthesis was reuced after the cells had fused into myotubes. The terminal addition of radioactive nucleotides into DNA strands occurred only to a minor extent, and the dilution of [3H] thymidine by intracellular nucleotide pools was shown not to be responsible for the observed difference in repair synthesis, Both the initial rate and the overall incorporation of [3H] thymidine were found to be 50% lower in the myotubes. 4NQO treatment of myoblasts and myotubes induced modifications in the DNA which were observed as single-strand breaks during alkaline sucrose sedimentation. After the myoblasts were allowed a post-treatment incubation, most of the single-strand breaks were not longer apparent. In contrast, a post-treatment incubation of myotubes did not change the extent of single-strand breakage seen. Both myoblasts and myotubes were equally effective in repairing single- strand breaks induced by X radiation. It would appear that when myoblasts fuse, a repair enzyme activity is lost, probably an endonuclease that recognizes one of the 4 NQO modifications of DNA. The result observed is a partial loss of repair synthetic ability and a complete loss of ability to remove the modification that appears as a single-strand break in alkali.     

Perturbation of DNA replication and cell cycle progression by commonly used [3H]thymidine labeling protocols.

The effect of tritiated thymidine incorporation on DNA replication was studied in Chinese hamster ovary cells. Rapidly eluting (small) DNA from cells labeled with 2 microCi of [3H]thymidine per ml (200 microCi/mmol) for 60 min matured to a large nonelutable size within approximately 2 to 4 h, as measured by the alkaline elution technique. However, DNA from cells exposed to 10 microCi of [3H]thymidine per ml (66 microCi/mmol) was more rapidly eluting initially and did not mature to a nonelutable size during subsequent incubation. Semiconservative DNA replication measured by cesium chloride gradient analysis of bromodeoxyuridine-substituted DNA was also found to be affected by the final specific activity of the [3H]thymidine used in the labeling protocol. Dramatic cell cycle perturbations accompanied these effects on DNA replication, suggesting that labeling protocols commonly used to study DNA metabolism produce aberrant DNA replication and subsequent cell cycle perturbations.     

Correlation between the presence of T-antigen and the reinitiation of host DNA synthesis in senescent human diploid fibroblasts after SV40 infection

Senescent human diploid fibroblasts, TIG-1, had labelling indices of about 0.5-3% when labelled with [3H]thymidine for 3 days in fresh medium containing 10% fetal bovine serum. When these cells were infected with SV40, the percentage of nuclei incorporating [3H]thymidine increased by about 10-fold. The frequency of T-antigen-positive cells and that of [3H]thymidine-incorporating cells were almost the same. About 80% of T-antigen-positive cells were also positive to incorporation of [3H]thymidine, and the same result was obtained in infected young cells. These results indicated that senescent human diploid cells which are brought to synthesize T-antigen always initiate DNA synthesis as young cells do. The characteristics of senescent cells as compared with younger cells was low incidence of T-antigen-positive cells after infection. The basis of low susceptibility of senescent cells to initiate DNA synthesis by SV40 infection thus seems to be concerned with an event after the adsorption of virus, but before the synthesis of a detectable amount of T-antigen.     

Inhibition of isoproterenol-induced DNA and RNA synthesis in rat parotid and pancreas following removal of submandibular-sublingual glands

Summary [³H] thymidine incorporation into DNA of the parotid (PA) gland of adult and 20-day-old rats and into DNA of the pancreas (PANC) of 20-day-old rats was increased markedly following a 2-day regimen of isoproterenol (ISO) administration. However, when the submandibular-sublingual (SM-SL) glands had been removed just prior to initiation of the ISO injections, the [³H] thymidine incorporation into PA and PANC was inhibited, and cpm/mg protein of these organs was even lower than that of organs of untreated rats with SM-SL glands present. Removal of the PA glands just prior to initiation of the ISO regimen had no effect on the ISO-induced [³H] thymidine incorporation into DNA of PANC but partially inhibited that of the submandibular (SM) gland. It is suggested that the inhibitory effects on DNA and RNA synthesis that follow removal of SM-SL glands are attributable to the growth factors (epidermal growth factor and nerve growth factor) found in the rat SM gland. These factors appear to regulate normal DNA synthetic activity of exocrine glands as well as ₁-adrenoceptor mediated DNA synthesis. Cellular hypertrophy induced by the ISO was less markedly affected by absence of the SM glands, but a partial inhibition of [³H] uridine incorporation into RNA of PA of adult rats also occurred when SM-SL glands were removed prior to initiation of the ISO-regimen.     

Repair of benzo[a]pyrene-initiated DNA damage in human cells requires activation of DNA polymerase alpha

Normal human fibroblasts treated with r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) yielded DNA polymerase alpha with elevated levels of activity, incorporated [3H]thymidine as a function of unscheduled DNA synthesis, and exhibited restoration of normal DNA-strand length as a function of unscheduled DNA synthesis. Lipoprotein-deficient fibroblasts treated with BPDE did not show elevated levels of DNA polymerase alpha activity, exhibited minimal [3H]thymidine incorporation, and had fragmented DNA after 24 h of repair in the absence of lipoprotein or phosphatidylinositol supplementation. When DNA polymerase beta activity was inhibited, cells with normal lipoprotein uptake exhibited [3H]thymidine incorporation into BPDE-damaged DNA but did not show an increase in DNA-strand length. DNA polymerase alpha activity and [3H]thymidine incorporation in lipoprotein-deficient fibroblasts increased to normal levels when the cells were permeabilized and low-density lipoproteins or phosphatidylinositol were introduced into the cells. DNA polymerase alpha isolated from normal human fibroblasts, but not from lipoprotein-deficient fibroblasts, showed increased specific activity after the cells were treated with BPDE. When BPDE-treated lipoprotein-deficient fibroblasts were permeabilized and 32P-ATP was introduced into the cells along with lipoproteins, 32P-labeled DNA polymerase alpha with significantly increased specific activity was isolated from the cells. These data suggest that treatment of human fibroblasts with BPDE initiates unscheduled DNA synthesis, as a function of DNA excision repair, which is correlated with increased activity of DNA polymerase alpha, and that increased DNA polymerase alpha activity may be correlated with phosphorylation of the enzyme in a reaction that is stimulated by low-density lipoprotein or by the lipoprotein component, phosphatidylinositol.     

Phenotypic conversion of TK-deficient cells following electroporation of functional TK enzyme.

The ability to phenotypically rescue a mutant (Rat-3, thymidine kinase-deficient) cell line by electroporation of functional TK enzyme has been investigated. Extracts of electroporated cells showed a 35-fold increase in TK enzyme levels under conditions where greater than 90% of the cells remained viable. The electroporated enzyme was intracellular, as demonstrated by the fact that cells were able to utilize exogenous [3H]thymidine for DNA synthesis. By in situ autoradiography, 82% of electroporated cells contained functional enzyme and incorporated [3H]thymidine into DNA. Thus, this technique can efficiently provide a missing metabolic function to cultured mammalian cells.     

DNA-synthesizing cells in oral epithelium have a range of cell cycle durations: evidence from double-labelling studies using tritiated thymidine and bromodeoxyuridine

Mouse tongue epithelium is characterized by a circadian variation in the number of DNA-synthesizing cells (labelling index, LI). Cells undergoing DNA synthesis were labelled with tritiated thymidine [( 3H]TdR) at 0300 (peak LI) or 1200 h (low LI). The fate of these cells was assessed by injecting animals with bromodeoxyuridine (BrdU) at intervals from 12-48 h after [3H]TdR, to follow them from one cell cycle to the next. Labelling was revealed by combining [3H]TdR autoradiography with immunoperoxidase detection of BrdU in the same sections. A single peak in the appearance of double-labelled cells was seen at 44 h, if [3H]TdR was given at 1200 h; following [3H]TdR at 0300 h, a peak of double labelling was seen at 48 h with the possibility of smaller peaks at 24 h and 36 h. These results show that the 24 h periodicity in LI in this tissue is associated with a predominant cell cycle duration of 44-48 h, but that a few cells cycle more quickly. Double labelling with [3H]TdR and BrdU provides a useful method for establishing cell cycle duration by labelling S-phase cells in successive cell cycles.