RECYCLING OF RESTING CELLS IN THE JB-1 ASCITES TUMOUR AFTER TREATMENT WITH 1-β-D-ARABINOFURANOSYLCYTOSINE (Ara-C)

Resting cells in tumours present a major problem in cancer chemotherapy. In the plateau phase of growth of the murine JB-1 ascites tumour (i.e. 10 days after 2–5 × 10⁶ cells i.p.) large fractions of non-cycling cells with G₁ and G₂ DNA content (Q₁ and Q₂ cells) are present, and the fate of these resting cells was investigated after treatment with l-β-d-arabinofuranosylcytosine (Ara-C). The experimental work consisted of growth curves, percentage of labelled mitoses curves after continuous labelling with ³H-TdR, and cytophotometric determination of single-cell DNA content in unlabelled tumour cells. Treatment with an i.p. single injection of Ara-C 200 mg/kg in the plateau JB-1 tumour resulted in a significant reduction in the number of tumour cells 1 and 2 days later as compared with untreated controls, while no difference in the number of tumour cells was observed after 3 days. In tumours prelabelled with ³H-TdR 24 hr before Ara-C treatment, a significant decrease in the percentage of labelled mitoses was observed 6–8 hr later followed by a return to the initial value after 12 hr, and a new pronounced fall from 20 hr after Ara-C. The second fall in the percentage of labelled mitoses disappeared when the labelling with ³H-TdR was continued also after Ara-C treatment. Cytophotometry of unlabelled tumour cells prelabelled for 24 hr with ³H-TdR before Ara-C treatment showed 20 hr after Ara-C a pronounced decrease in the fraction of Q_t cells paralleled by an increase in the fraction of unlabelled cells with S DNA content. These results indicate recycling of resting cells first with G₂ and later with G_x DNA content, which contribute to the regrowth of the tumours.     

Querscheibenspezifische Unterschiede in der3H-Thymidin-Markierung während der Larvenentwicklung vonChironomus thummi piger

In salivary gland chromosome II ofChironomus, region A2j-A3(d) replicates during the whole replication cycle.³H-thymidine is incorporated in this region for a longer time than in bands of greater DNA values (Hägele, 1970). However, no extra DNA is accumulated in A2j-A3(d). Therefore it was supposed that in addition to the duplication of structural DNA an extra DNA is synthesized which immediately disappears from the chromosome. In this report the attempt was made to test this hypothesis. — Using³H-thymidine, the autoradiographic patterns have been studied which occur in the salivary gland chromosomes II ofChironomus thummi piger at the end of a replication step. The probable order of their sequence has been established. At the mid phase of a replication step region A2j-A3(d) and a certain number of definite bands are labelled whereas at the very end of DNA synthesis only A2j-A3(d) shows labelling. It is demonstrated that this region replicates for a longer time than regions containing up to 3.8 times more DNA. Moreover in most cells³H-thymidine is incorporated in region A2j-A3(d) at the end of synthesis at a higher rate than in late replicating bands. In this region there exists a considerable difference in relative grain density within the same phase of a replication step. This difference cannot be found in other bands studied. — These labelling patterns occur in chromosomes of both young larvae (8–9 days old) and prepupae (15–17 days old) if the larvae are prepared immediately after incubation in the isotope. — However, if young larvae are incubated in³H-thymidine and then develop to prepupae in water free of isotope region, A2j-A3(d) is unlabelled at the end of a replication step in half of the cells studied. In the other half this region shows labelling but the relative grain density is markedly reduced. The labelling pattern of other bands is not changed. Therefore loss of radioactive DNA in A2j-A3(d) is of real occurrence. — This loss probably takes place within the replication steps 1 or 2 between young larvae and praepupae. In these replications the structural DNA and the extra DNA, newly synthesized in A2j-A3(d), are unlabelled. The extra DNA disappears immediately from the chromosome. If, by chance, an exchange takes place between newly synthesized unlabelled DNA chains of extra DNA and old labelled DNA, then loss of radioactive DNA would be the result.     

Recycling of resting cells in the JB-1 ascites tumour after treatment with 1-beta-D-arabinofuranosylcytosine (ara-C).

Resting cells in tumours present a major problem in cancer chemotherapy. In the plateau phase of grwoth of the murine JB-1 ascites tumour (i.e. 10 days after 2-5 X 10(6) cells i.p.) large fractions of non-cycling cells with G1 and G2 DNA content (Q1 and Q2 cells) are present, and the fate of these resting cells was investigated after treatment with 1-beta-D-arabinofuranosylcytosine (Ara-C).The experimental work of growth curves, percentage of labelled mitoses curves after continuous labelling with 3H-TdR, and cytophotometric determination of single-cell DNA content in unlabelled tumour cells. Treatment with an i.p. single injection of Ara-C 200 mg/kg in the plateau JB-1 tumour resulted in a significant reduction in the number of tumour cells 1 and 2 days later as compared with untreated controls, while no difference in the number of tumour cells was observed after 3 days. In tumours prelabelled with 3H-TdR 24 hr before Ara-C treatment, a significant decrease in the percentage of labelled mitoses was observed 6-8 hr later followed by a return to the initial value after 12 hr, and a new pronounced fall from 20 hr after Ara-C. The second fall in the percentage of labelled mitoses disappeared when the labelling with 3H-TdR was continued also after Ara-C treatment. Cytophotometry of unlabelled tumour cells prelabelled for 24 hr with 3H-TdR before Ara-C treatment showed 20 hr after Ara-C a pronounced decrease in the fraction of Q1 cells paralleled by an increase in the fraction of unlabelled cells with S DNA content. The results indicate recycling of resting cells first with G2 and later with G1 DNA content, which contribute to the regrowth of the tumours.     

Late DNA replicaion of X chromosomes in female and pseudofemale cells of Microtus agrestis.

The late replication pattern of the short arms of the X chromosomes of Microtus agrestis was studied in female cells and in cells with 2 X chromosomes of male origin by means of the BUdR-Giemsa technique and of 3H-thymidine labelling. The light absorption of Giemsa stained chromosome sections which were unifilarly substituted with BUdR (labelled), was found to be 59.2% of that of unlabelled chromosomes. In female cells, asynchrony of DNA replication of both X chromosomes indicated the presence of facultative heterochromatin in the X2 and euchromatin in the X1. In the male cells only euchromatic X chromosomes were observed in diploid XX and XO cells as well as in triploid XXY, XX and XO cells. The results show that inactivation of an X chromosone in vitro, in cells with more than one originally active X chromosome does not occur even after a culture duration of several years.     

Asynchrony of DNA replication in the chromosomes of Luzula

Seedlings of Luzula purpurea (2n=6) were placed in contact with H³-thymidine for 30 minutes. After removal of the isotope the roots and leaf primordia were fixed at intervals between 0 and 14 hours. The percentage of labelled mitoses follows a very close curve in roots and leaf primordia. In both tissues the value of G₂ is approximately 3 to 4 hours and of S circa 8 hours. DNA replication in the chromosomes of L. purpurea is asynchronous. The discontinuous DNA synthesis discloses that Luzula chromosomes are composed of many segments replicating independently of each other. The results support a polycentric rather than a completely diffuse kinetochore system in this species.     

Ribosomal RNA gene amplification by rolling circles

Previous work has raised the possibility that gene amplification in Xenopus laevis oocytes involves a rolling circle intermediate (Hourcade et al., 1973a,b). We have combined electron microscopy with autoradiography in order to examine the structure of replicating ribosomal DNA molecules. The frequency of lariats (the presumptive rolling circles) in unlabelled ribosomal DNA is about 1%. If only labelled molecules are scored after a six-hour pulse with radioactively labelled DNA precursors, the proportion of lariats increases to about 8.5%. After pulses of two hours or less, the frequency rises still further to about 18%. In pulse and pulse-chase experiments, the lariats display a labelling pattern that is consistent with a rolling circle model: (1) as the pulse length is increased the labelled region in the tail grows from the replication fork towards the free end of the tail; (2) after a pulse-chase the labelled region is displaced towards the free end of the tail and no label remains associated with the lariat circle. The frequency of labelled free circles is lower by 80% in pulse-chased DNA than in DNA that has not been chased. This suggests that most of the radioactive circles are derived from broken rolling circles. Cyclization of lariat tails could account for the remaining labelled circles.     

Influence of growth hormone and thyroxine on cell kinetics in the proximal tibial growth plate of Snell dwarf mice

In Snell dwarf mice, the influence of short-term treatment with human growth hormone (hGH) or thyroxine on the proliferative and sulphation activity of the proximal tibial growth plate was studied. By autoradiographic methods, the [3H]methylthymidine incorporation after a single injection was measured, after 2 hr incorporation time. The labelling index was calculated and the number of labelled mitoses was counted. In addition, the distribution of the labelled nuclei over the proliferating and degenerating zones was determined by continuous labelling for 25 and 73 hr. In untreated dwarf mice after [3H]-methylthymidine administration, the number of labelled nuclei in the growth plate is low. Labelling occurs, as expected, mainly in the cells of the proliferative zones. The number of labelled nuclei in control dwarf mice was similar after 25 and 73 hr continuous labelling. This suggests that many cells are in a resting G0 or prolonged G1 phase. Both hGH and T4 treatment induce a significant increase of the number of labelled nuclei per growth plate and of the number of mitoses. Since hormonal treatment induces a small number of mitoses after 2 hr incorporation of the label, the minimal G2 phase of the cell cycle is less than 2 hr. In addition, treatment with hGH and T4 stimulates chondrocytes in the zone of proliferative and hypertrophic cells to actively incorporate [35S]-sulphate.     

Replication in Drosophila chromosomes XIII. Comparison of late replicating sites in two polytene cell types in D. hydei

We have compared the temporal order of completion of replication of specific sites of X and 2nd chromosomes in two polytene cell types of D. hydei by examining the patterns of autoradiographic labelling in ³H-thymidine pulse (10 min) labelled salivary glands and gastric ceaca of mid 3rd instar larvae. Present results are in agreement with our earlier finding in D. nasuta (Lakhotia & Tiwari, 1984, Chromosoma, 89: 212–217 that in spites of a general similarity in the cytological identity of independently replicating sites in the two polytene cell types, their temporal programme of replication varies in different tissues. This may be related to differential gene activity patterns and polytene organization in the different cell types.     

Studies of multipolar mitoses in euploid tissue cultures

In tissue cultures of male Microtus agrestis, diploid mitoses with two X or two Y chromosomes were found. For identifiying the sex chromosomes in nonhypotonioally treated mitoses, the asynchrony of DNA replication of the sex chromosomes of both sexes was used. The constitutive heterochromatin of Y replicates later in the S period than X, and X₂ of the female replicates later than X₁. Autoradiographic studies of tetraploid tripolar mitoses showed that the diploid daughter nuclei contain either XX or YY in the male; in the female, X₁X₂ daughter nuclei were found less frequently than X₁X₁ and X₂X₂ cells.     

Comparative study of human chromosome replication in primary cultures of embryonic fibroblasts and in cultures of peripheral blood leucocytes

By autoradiography with ³H-thymidine and ³H-deoxycytidine it is shown that chromosomes 1 and 16 in cultures of embryonic fibroblasts at the termination of the S period synthesise AT- and GC-rich DNA at different rats: in both chromosomes the labelling of AT-bases is more intensive. In leucocyte cultures both nucleotide pairs label equally in these chromosomes. Chromosomes 2, 3, 4–5 and 21–22 are labelled equally in both cultures with respect to AT-and GC-pairs. Fibroblasts and leucocytes differ in the relative intensity of DNA synthesis at the end of the S period: chromosomes 1,16 and 21–22 contain more label in the case of fibroblasts (chromosome 1 solely due to AT-pairs) and chromosome 4–5 in the case of leucocytes. Analysis of distribution of late label along chromosome 1 showed that in fibroblast cultures the pericentromeric regions of both arms are labelled more intensively in respect to both nucleotide pairs than in leucocyte cultures. Both in fibroblast and leucocyte cultures no significant distinctions in the distribution of AT-and GC-pairs along chromosome 2 were established. In fibroblast cultures the pericentromeric regions of both arms of chromosome 3 are labelled more intensively than other regions. In leucocyte cultures the pericentromeric region of the short arm of this chromosome is labelled with the same intensively as in fibroblasts, whereas in the pericentromeric region of the long arm the intensity of incorporation of labelled synthesis precursors decreases. — Analysis of results obtained in the present study together with data of previous studied (Slesinger et al., 1974; Lozovskaya et al., 1976; Lozovskaya et al., 1977) shows that differences between the two types of cells in the intensity of late ³H-thymidine labelling in the C-heterochromatin regions of chromosomes 1 and 16 may be explained both by variation of replication time in leucocytes as compared with fibroblasts and by variation of the content of AT- rich DNA. Differences observed in other chromosomes are probably due to different times of replication of these chromosomes in leucocytes and fibroblasts. — Thus, the process of cell system differentiation involves not only differential activity of the genome (the main mechanism) that is connected with differences in the replication time of chromosomes and of their regions but also variation of the quantity of genetic material.     

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.     

Autoradiographic study of the effect of 5-Fluorodeoxyuridine on barley chromosomes

FUdR 10^?4 M was applied together with [³H] TdR on the growing barley embryos cultivated in the nutrient solution. Samples were fixed 1–4 h after the onset of the treatment and microautoradiograms were prepared. All the mitoses were unlabelled, while more than 150 autoradiographic grains were found above labelled interphase nuclei; no cells that terminated S phase at the onset of the treatment passed through entire G₂ and reached mitosis during the 4 h duration of the treatment. Chromosomal fragments in anaphases appeared the first hour after the onset of the treatment. Their frequency increased from the first to the second hour and remained almost constant from the second to the fourth hour. FUdR induces chromosomal fragments in barley root meristems also in G₂ phase of the mitotic cycle.     

The effect of supernumerary chromosomes on meiosis in Puschkinia libanotica (Liliaceae)

The presence of the partly heterochromatic supernumerary chromosomes in pollen mother cells of Puschkinia libanotica raises the chiasma frequency in the A chromosome bivalents. The pattern of chiasma distribution along each of the five A bivalents was related to the DNA labelling pattern of mitotic chromosomes. Regions that showed heavy labelling at the end of the DNA synthetic phase had fewer chiasmata than lightly labelled regions. As this relation is the opposite to that found by Rees and Evans in another species we regard any correlation between labelling pattern and chiasma distribution as fortuitous.     

Nitrogen and phosphorus cycling in relation to stand age of Eucalyptus regnans F. Muell

Lupins, canola, ryegrass and wheat fertilized with Na₂ ³⁵SO₄ and either ¹⁵NH₄Cl or K¹⁵NO₃(N:S=10:1), were grown in the field in unconfined microplots, and the sources of N and S (fertilizer, soil, atmosphere, seed) in plant tops during crop development were estimated. Modelled estimates of the proportion of lupin N derived from the atmosphere, which were obtained independently of reference plants, were used to calculate the proportion of lupin N derived from the soil. Total uptake of N and S and uptake of labelled N and S increased during crop development. Total uptake of S by canola was higher than lupins, but labelled S uptake by lupins exceeded uptake by canola. The form of N applied had no effect on uptake of labelled and unlabelled forms of N or S. Ratios of labelled to unlabelled S and ratios of labelled to unlabelled N derived from soil sources decreased during growth, and were less for S than for N for each crop at each sampling time. Although ratios of labelled to unlabelled soil-derived N were similar between crops at 155, 176 and 190 days after sowing, ratios of labelled to unlabelled S for lupins were higher than for the reference crops and declined during this period. The ratios of labelled to unlabelled S in lupins and the reference plants therefore bore no relationship either to ratios of labelled to unlabelled soil-derived N in the plants, or to total S uptake by the plants. Therefore the hypothesis that equal ratios of labelled N to unlabelled soil-derived N in legumes (Rleg) and reference plants (Rref) would be indicated by equal ratios of labelled to unlabelled S was not supported by the data. The results therefore show that the accuracy of reference plant-derived values of Rleg cannot be evaluated by labelling with ³⁵S.     

Replication in Drosophila chromosomes

Prolongation of larval life in Drosophila melanogaster, by growing wild type larvae at lower temperature, or in animals carrying the X-linked mutation giant is known to result in a greater proportion of nuclei in salivary glands showing the highest level of polyteny. We have examined by autoradiography the patterns of ³H-thymidine incorporation during 10 min or 1 min pulses in salivary gland polytene chromosomes of older giant larvae and of wild type late third instar larvae of D. melanogaster grown since hatching either at 24 ° C or at 10 ° C. The various patterns of labelling and their relative frequencies are generally similar in glands from the warm-(24 ° C) or cold (10 ° C)-reared wild type larvae, except the interband (IB) labelling patterns which are very frequent in the later group but rare in the former. The IB type labelled nuclei in cold-reared wild type larvae show labelling ranging from only a few puffs/interbands labelled to nearly all puffs/interbands labelled. In warm-reared wild type larvae, very low labelled IB patterns are not seen. In older giant larvae, the ³H-thymidine labelling patterns are in most respects similar to those seen in cold-reared wild type larvae. In 1 min pulsed preparations from all larvae, the IB patterns are relatively more frequent than in corresponding 10 min pulsed preparations. No nuclei with the continuous (2C or 3C) type of labelling pattern, with all bands and interbands/puffs labelled, were seen in 1 min pulsed preparations from cold-reared wild type or in giant larvae, and only a few nuclei in 1 min pulsed preparations from warm-reared wild type larvae exhibited the 2C labelling pattern. Analysis of silver grain density on specific late replicating sites in late discontinuous (1D) type labelled nuclei suggests that the rate of DNA synthesis per chromosomal site is not different at the two developmental temperatures. It is suggested that correlated with the prolongation of larval life under cold-rearing conditions or in giant larvae, the polytene replication cycles are also prolonged. It is further suggested that the polytene S-period in these larvae is longer due to a considerable asynchrony in the initiation and termination of replication of different sites during a replication cycle.     

Proceedings: Heterochromatin and chromosome aberrations: a comparative study of normal and tumour cells of mouse with various distributions of heterochromatin.

Seedlings of Crepis capillaris were irradiated after pulse-labelling with tritiated thymidine ([³H]TdR), and both chromosomal aberrations and presence of silver grains were recorded in the same metaphase cells at various intervals throughout the whole mitotic cycle. The following results were obtained: (a) irradiated roots were homogeneous with respect to the number of aberrations, and heterogenous with respect to labelling index (LI); (b) time-effect curves for labelled (L) and unlabelled (U) cells showed no significant difference from one another; (c) no significant quantitative difference of aberration spectra produced in S and G₂ stages was found. These results support the view that the major factor which determines both quantitative and qualitative variation in the production of chromosomal aberrations by radiation is the time lapse between irradiation and fixation rather than relation of the time of irradiation to the time of DNA synthesis. In addition, it was found that labelling with [³H]TdR modifies the effect of radiation on chromosomes.     

Labelling of human chromosomes with 3H-AMD

Summary This paper presents data on the pattern obtained by labelling in vitro with ³H-AMD human chromosomes from peripheral blood cultures. Preparations were divided into two experimental groups: 1. preparations labelled with ³H-AMD; 2. preparations treated with H₂SO₄ and labelled with ³H-AMD.The results show evidence of non-random grain distribution for some chromosomes and of differential intensity of labelling of chromosomes within some chromosome groups. Treatment with H₂SO₄ before labelling with ³H-AMD resulted in random distribution of grains along the chromosomes with disappearance of any labelling pattern. The same result was observed when preparations were stained with quinacrine dihydrochloride before labelling with ³H-AMD. These data suggest the existence of a differential distribution of proteins along the chromosomes.

---

Zusammenfassung In der vorliegenden Arbeit wird die in vitro-Markierung von menschlichen Chromosomen mit ³H-Aktinomycin D (AMD) in Kulturen des peripheren Blutes beschrieben. Es handelt sich um zwei Versuchsgruppen: 1. Markierung der Präparationen mit ³H-AMD; 2. Markierung der Präparationen mit ³H-AMD nach Vorbehandlung mit Schwefelsäure.Bei diesen Versuchen ergaben sich Hinweise auf eine nichtzufallsmäßige Kornverteilung auf einigen Chromosomen und auf differentielle Markierungsmuster in den Chromosomen einiger Chromosomengruppen. Diese Markierungsmuster wurden durch Vorbehandlung mit Schwefelsäure zerstört. Das gleiche Resultat wurde erhalten, wenn die Präparate vor der Markierung mit Chinakrin-Dihydrochlorid gefärbt wurden. Diese Ergebnisse lassen vermuten, daß die Proteine auf den Chromosomen ungleichmäßig verteilt sind.

     

Early replicating DNA in heterochromatin ofPlagiochila ovalifolia (Liverworts)

The timing of DNA replication of heterochromatin in malePlagiochila ovalifolia was investigated by the use of³H-thymidine autoradiography. The estimated duration of the mitotic cycle was as follows: S period, 19 hr: G₂+prophase, 10 hr; G₁+meta-, ana-, telophase, 5 hr; total mitotic cycle, 34 hr. The first appearance of silver grains over the chromosomes was observed at 8 hr after the beginning of pulse labelling at which time the silver grains were only over the euchromatic regions, not over the heterochromatic regions. This labelling pattern was also observed at 10 to 15 hr. The heterochromatic regions having more grains than the euchromatic regions were observed at 20 to 25 hr. These results show that the DNA of the heterochromatin of this species is replicated earlier than the euchromatin.     

Chromosomal localizations by in situ hybridization of the repetitious human DNA families and evidence of their satellite DNA equivalents

Four of the five major repetitious human DNA families, have been mapped by the in situ hybridization technique at their T_OPT values. Two of the lighter density DNA families have autoradiographic grain patterns over heterochromatic chromosomal regions that resemble those of known satellite DNAs. The two heaviest density DNA families have autoradiographic grain patterns of middle repetitious DNAs, with all chromosomes showing labelling. Some evidence suggests that one of these DNA families is concentrated in certain chromosomal regions. Both DNA families exhibit biphasic T_OPT curves. The presence of two thermal stability classes of hybrids suggests sequence interspersion. By co-enrichment studies in Ag⁺-Cs₂SO₄ gradients, evidence suggests the origin of the three lightest density renaturated human DNA families to be satellites I, II and III.