Quantitating the frequency of initiation and cH-ras mutation in in situ N-methyl-N-nitrosourea-exposed rat mammary gland

Mammary carcinogenesis is a multistep process consisting minimally of initiation and promotion/progression stages. The rate-limiting stage in the carcinogenesis process is undetermined but can in part be addressed by estimating the frequency of initiation, a heritable early event. Here, we use an in vivo limiting dilution transplantation assay to estimate initiation frequency in a rat mammary epithelial stem-like cell population that was exposed in situ to 50 mg/kg N-methyl-N-nitrosourea (NMU) administered i.v. We estimate that this dose resulted in the killing of 65% of exposed mammary cells. Known numbers of cells surviving NMU exposure were grafted into fat-pads of recipient rats in which the cells grew and differentiated into structurally and functionally normal mammary glands. Recipient rats were hormonally manipulated to provide maximal promotion of initiated cells. Mammary carcinomas developing at graft sites were quantitated over a 2-year period. Based on these results, we estimate that at least 1 surviving NMU-exposed mammary cell in 7,200 was initiated. Seventeen % of these graft site carcinomas had an activated H-ras oncogene with a G to A mutation in codon 12. This suggests that at least 1 mammary cell in 43,000 was mutated in this fashion by in situ exposure to NMU. These data suggest that cH-ras represents approximately 1 of 5 of the initiation events produced by NMU exposure of rat mammary glands.     

Clonogenic cells and rat mammary cancer: effects of hormones, X rays, and fission neutrons

On Day 0, young adult female F344 rats were adrenalectomized and intrasplenically implanted with a pituitary gland and capsule containing estrone. All were thereafter given 2.5 mg deoxycorticosterone per week and the choice of saline or tap water. This treatment yields high prolactin levels and glucocorticoid deficiency (Prl+/Glc-). On Day +48, total recoverable mammary DNA was increased by more than sevenfold, tritiated thymidine uptake by nearly fourfold, and total mammary clonogens by about fivefold. Irradiation with 4, 40, and 80 cGy X rays on Day +48 increased total mammary carcinomas per rat day at risk linearly with dose, and 40 and 80 cGy significantly decreased first carcinoma latency. A dose of 40 cGy X rays on Day -1 yielded tumor latencies and frequencies insignificantly different from unirradiated controls and significantly different from the dose on Day +48. Total carcinomas per rat day at risk were better fit by a function of dose to the power 0.4 than by a linear function after exposure to 1, 10, and 20 cGy fission neutrons, and 10 and 20 cGy significantly shortened the time to appearance of the first cancer. In contrast to results with X rays, 10 cGy neutrons on Day -1 yielded tumor frequencies and latencies insignificantly different from 10 cGy neutrons on Day +48. The carcinogenic action of X rays was thus influenced by total clonogen numbers and/or proliferation rates; that of neutrons was not.     

Tumour bed effect: hypoxic fraction of tumours growing in preirradiated beds

The reduction in tumour growth rate seen when tumours are implanted into preirradiated sites, the tumour bed effect (TBE), is believed to be due to radiation damage to vascular stroma, leading to defective angiogenesis in the tumour. The present work examined whether or not the functional inadequacy of irradiated stroma was accompanied by an increased hypoxic fraction in tumours growing in irradiated beds. Mouse flank skin was given 0 or 20 Gy X-rays and RIF-1 fibrosarcoma cells were implanted i.d. into the centre of the treatment field one week later. Tumours of 200 mm3 were irradiated under clamped or unclamped conditions and the hypoxic fraction measured from the displacement of the corresponding survival curves, assayed in vitro. Results indicated a small increase in the hypoxic fraction. Averaging values from three independent experiments, the percentage of hypoxic cells increased from 2.5 per cent for cells in tumours growing in unirradiated beds to 4.6 per cent for those from tumours in beds given 20 Gy. Thus an irradiated vascular bed is still to some extent able to maintain the proportion of oxic: hypoxic tumour cells found in tumours growing in unirradiated beds, despite manifest changes in tumour necrosis and growth rate.     

Identification of a plasmid-coded protein required for initiation of ColE2 DNA replication.

The product of the rep gene of ColE2 is required for initiation of ColE2 DNA replication. The rep gene was placed under the control of the promoters, PL and PR, and the heat-labile cl857 repressor of bacteriophage lambda. The Rep protein was identified as a 35 Kd protein by the maxicell method in combination with heat-induced expression. The protein was efficiently expressed from these promoters in unirradiated cells and accumulated up to a few per cent of the total cellular proteins. It was partially purified (about 80% pure) and its properties examined. The amino acid sequence of the amino terminal portion of the partially purified protein agreed well with that predicted from the nucleotide sequence of the rep gene. One of the characteristic features of the rep gene is frequent usage of rare codons, especially those for arginine. The protein specifically stimulated replication of ColE2 DNA but not that of ColE3 DNA in crude cell extracts of Escherichia coli. Specific binding of the protein to plasmid DNA containing the origin region of ColE2 was demonstrated by the filter binding method. Neither endonuclease activity nor topoisomerase activity was detected by using ColE2 DNA.     

Survival curves for normal-tissue clonogens: a comparison of assessments using in vitro, transplantation, or in situ techniques

A survey of survival curves in the literature, for clonogenic cells (clonogens) in normal tissues, highlights the following features: the sensitivity of some human and dog clonogens apparently is greater than that of their counterparts in mice and sheep, assessed in vitro. However, this should be interpreted with caution because of the possibility of cell selection and the ability to modify sensitivity markedly in some systems by variations in growth conditions; extrapolation numbers are in general higher when assessed in vivo than in vitro. This is due partly to the lack of measurements of repair of potentially-lethal damage using many assays in vitro. This feature increases the extrapolation number when measured using transplantation assays in vivo; epithelial clonogens in vivo demonstrate a remarkable similarity in sensitivity between tissues. The range is similar for clonogens assayed in situ or by transplantation, and this argues against the possibility that a resistant subpopulation may be selected in most assays in situ. It is emphasized from the comparisons that caution must be exercised in extrapolating results, obtained for clonogens assayed in vitro or by transplantation in vivo, to the situation in situ.     

MEASUREMENTS OF SOME CELLULAR CHANGES DURING THE FIXATION OF AMPHIBIAN ERYTHROCYTES WITH OSMIUM TETROXIDE SOLUTIONS

On average, 15 per cent of the total haemoglobin present in the blood of the newt Triturus cristatus was extracted during 45 minutes of fixation in Palade-Caulfield fixative. This extraction was reduced with fixatives buffered at pH 6.2 instead of pH 7.4. The addition of Ca⁺⁺ ions to a final concentration of 0.01 M in the fixative completely suppressed haemoglobin extraction. The effect of the pH, and the presence or absence of Ca⁺⁺ ions in the fixative, on the rate of haemoglobin extraction has been determined. During Palade-Caulfield fixation the average projected area of newt erythrocytes increased by 37 per cent, and after dehydration and embedding in Epon the average area was 25 per cent greater than that of the unfixed cell. Fixatives buffered at pH 6.2 and containing 0.01 M Ca⁺⁺ ions caused cellular shrinkage, with the average projected area decreasing by 10 per cent in the fixative. This shrinkage continued during dehydration, and the final average area of the erythrocytes in Epon was 26 per cent less than that of the unfixed cells. Similar measurements with erythrocytes of Amphiuma tridactylum showed that after Palade-Caulfield fixation the average cellular area was increased by 45 per cent, and after dehydration and embedding in Araldite it was 36 per cent greater than that of the unfixed cell. The average nuclear area increased by 35 per cent during fixation but after embedding it was 26 per cent greater than that of the unfixed nuclei. With a fixative at pH 6.2 containing 0.01 M Ca⁺⁺ ions, both the nucleus and the whole cell shrank during fixation. The nuclear area decreased by 20 per cent and the cellular area by 22 per cent. After dehydration and embedding in Araldite, the average nuclear area had decreased by 35 per cent and the cellular area by 40 per cent. It has been shown that OsO₄ fixation lowers the isoelectric points of haemoglobins and other proteins. This finding has been used in the interpretation of the observed cellular changes resulting from fixation.     

Contributions of cytoplasmic factors to in vitro cellular senescence.

Mass populations of normal human lung fibroblasts were enucleated by centrifugation at greater than or equal to 25,000 g in 4 mug/ml cytochalasin B. The 1 per cent of cells that did not enucleate where rendered nonviable by treatment with mitomycin C. Whole cells were poisoned with a 99 per cent lethal dose of the sulfhydryl reagent iodoacetate. The washed cells were then mixed with the anucleate cytoplasms, fused with inactivated Sendai virus, and planted in rotenone for 20 hours. Whereas normal cells are able to survive this rotenone treatment, the 1 per cent surviving iodoacetate-treated cells cannot withstand this additional stress. However, iodoacetate treated cells that fuse to untreated cytoplasms receive sufficient amounts of active enzymes to allow them to survive. Since this selective system does not rely on using enzymatic mutants, it should permit the selection of hybrids between anucleate cytoplasms and any type of whole cell. Cytoplasmic hybrids were cultured in order to determine their proliferative capacity. The life-spans of cytoplasmic hybrids between young and old cells were compared to those of young/young and old/old controls. Cytoplasmic factors do not appear to control in vitro cellular senescence.     

Clonogen number and radiosensitivity in rat thyroid follicles

A technique has been developed for transplanting whole thyroid follicles into the fat pads of recipient thyroidectomized rats to assess the ability of the follicular cells to proliferate and form colonies, i.e., clusters of new follicles. Of the transplanted follicles, 80-90% formed follicle clusters, indicative of the presence of transplanted follicles not containing colony-forming cells (clonogens) or some reproducible degree of transplantation trauma. The initial number of clonogens per regenerative follicle was calculated from data from split-dose experiments to be 3.3 +/- 1.5, and their sensitivity was characterized by a D0 value of 350 +/- 95 cGy. Three clonogens among about 80 epithelial cells per regenerative follicle and the 10-20% of nonregenerative follicles represent an overall colony-forming efficiency of about 3%. This is similar to the value of 2-3% reported by others using single-cell transplantation techniques.     

Influence of seed moisture content and post-irradiation hydration temperature on the kinetics of reactivity towards oxygen or decay of oxygen-sensitive sites.

The rate of development of post-irradiation oxygen-dependent damage when oxygen is available, and its rate of elimination when seeds are first post-hydrated in oxygen-free water prior to their transfer to oxygenated water, was studied in barley seeds of approximately 3 per cent, approximately 8 per cent and approximately 9 per cent moisture contents at 3 degrees C, 25 degrees C and 37 degrees C. The magnitude of oxic damage at a given dose (35 krad) decreases as the initial seed moisture content increases from approximately 3 per cent to approximately 9 per cent. Significant (P = 0.01) oxic damage is observed in seeds of all the three moisture contents at 3 degrees C and 25 degrees C; however, at 37 degrees C significant (P = 0.01) oxic damage is observed only in seeds of approximately 3 per cent and approximately 8 per cent moisture contents. The magnitude of oxic damage in seeds of a given moisture content remains unaltered following oxygenated post-hydration of seeds at 3 degrees C and 25 degrees C, but it registers a significant (P = 0.01) decrease if post-hydration in oxygenated water is carried out at 37 degrees C. The radiation-induced oxygen-sensitive (An) sites react with oxygen approximately 6 to 8 times faster as compared to their rate of decay in the absence of oxygen at both 3 degrees C and 25 degrees C; however, at 37 degrees C they react only approximately 3 to 4 times faster, in seeds of all the three moisture contents. Moreover, the initiation of the decay of An sites becomes evident much earlier in very dry (approximately 3 per cent moist) seeds than in relatively moist (approximately 8 per cent and approximately 9 per cent) seeds. It is also observed that this fraction of An sites which is capable of a very rapid rate of decay in the absence of oxygen is capable also of an even more rapid rate of reactivity towards oxygen.     

Study of possible biological transformation of kanamycin to amikacin]

For transformation of kanamycin A (Km) to amikacin (Ak) with acylating enzymes from B. circulans, a culture producing butirosin (Btn), cellular and acellular conversion systems and methods for chemical and biological identification of Km, Ak and Btn were developed. The level of conversion of Km to Ak in vivo and in vitro did not exceed 2 per cent.     

v-ras genes from Harvey and BALB murine sarcoma viruses can act as initiators of two-stage mouse skin carcinogenesis

Activated Harvey murine sarcoma virus ras genes were introduced into epidermal cells in vivo by direct application of retroviruses to mouse skin. Subsequent treatment with the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced benign papillomas, some of which progressed to invasive carcinomas. Initiation with virus was irreversible for at least 4 months, since TPA treatment after this latency period produced papillomas within 4 weeks. Analysis of viral integration sites showed that carcinomas are clonal in origin. Both papillomas and carcinomas express virus-specific ras mRNA and the viral form of ras P21 protein. The results show that activated ras genes can replace chemical carcinogens in initiation of mouse skin carcinogenesis. This system presents a novel approach to in vivo analysis of the biological role of oncogenes in epithelial tumorigenesis.     

The gastrointestinal syndrome and mucosal clonogenic cells: relationships between target cell sensitivities, LD50 and cell survival, and their modification by antibiotics

The sensitivity of the target cells responsible for the gastrointestinal syndrome in mice was deduced from the steepness of the dose-survival curve for mice assessed on Day 7 after irradiation. The D0 value was 1.25 +/- 0.22 Gy, virtually identical to the value of 1.23 +/- 0.08 measured for microcolony-forming cells (clonogens) over about the same range of dose in concurrent experiments. The survival of clonogens was similar when assayed in mice surviving to Days 3, 4, or 5, but clonogenic sensitivity was lower when assessed on Day 7. This was shown at one dose to be due largely to a selection of mice with high colony counts with only a small contribution from crypt budding. The LD50 for mice corresponded to a surviving fraction of crypts of about 0.35. An injection of 5 mg streptomycin sulphate ip daily for 5 days after irradiation increased the latent period by about 1 day, increased the LD50 by about 1.4 Gy, but did not significantly change the survival of clonogens. These studies are the first to test and satisfy the interpretation of a dose-response curve for animal survival in terms of "target cell" survival, where measurements of both are made over a similar range of dose in concurrent experiments.     

Iterated birth and death process as a model of radiation cell survival

The iterated birth and death process is defined as an n-fold iteration of a stochastic process consisting of the combination of instantaneous random killing of individuals in a certain population with a given survival probability s with a Markov birth and death process describing subsequent population dynamics. A long standing problem of computing the distribution of the number of clonogenic tumor cells surviving a fractionated radiation schedule consisting of n equal doses separated by equal time intervals tau is solved within the framework of iterated birth and death processes. For any initial tumor size i, an explicit formula for the distribution of the number M of surviving clonogens at moment tau after the end of treatment is found. It is shown that if i-->infinity and s-->0 so that is(n) tends to a finite positive limit, the distribution of random variable M converges to a probability distribution, and a formula for the latter is obtained. This result generalizes the classical theorem about the Poisson limit of a sequence of binomial distributions. The exact and limiting distributions are also found for the number of surviving clonogens immediately after the nth exposure. In this case, the limiting distribution turns out to be a Poisson distribution.     

A single serine residue at position 375 of VP16 is critical for complex assembly with Oct-1 and HCF and is a target of phosphorylation by casein kinase II.

We show that VP16 is phosphorylated by cellular kinases in vivo and in vitro and map the major sites of phosphorylation to be on serines towards the C-terminus, downstream of position 370 in both cases. Deletion of the acidic activation domain had no effect on phosphorylation, refining the sites to between position 370 and 411. Within VP16, the C-terminal boundary for complex formation with Oct-1 and HCF lies at position 388, and between 370 and 388 lies one serine, at position 375. This is a consensus casein kinase II (CKII) site and, using purified wild-type and mutant proteins, we show that it is the main CKII site in the body of the N-terminal complex-forming region. This site is also phosphorylated in nuclear extracts. Although other sites, mainly Ser411, are also phosphorylated by nuclear kinase(s), the single substitution of Ser375 to alanine abolishes CKII phosphorylation in vitro and virtually eliminates complex formation. This serine lies in a surface-exposed region of VP16 and, although complex formation is disrupted, other activities of the mutant are unaffected. Ser375 is also required in vivo where substitution to alanine abolishes transactivation, while replacement with threonine restores normal levels of activity.     

Nitrogen cycling in grasslands at Kanpur,India

Summary The present paper deals with the distribution of nitrogen in the different plant compartments and in the top 30 cm soil among the protected, semi-protected and open-grazed grasslands at Kanpur (26° 26 N latitude and 80° 22 E longitude).The protected site indicated greater nitrogen content (g N m^–2) in the aboveground and belowground plant parts as compared to those of semi-protected and open-grazed sites. Nitrogen content in the combined live and dead herbage varied from 2.6 to 53.5 g m^–2 in protected community, 1.6 to 27.6 g m^–2 in semi-protected and 0.9 to 17.4 g m^–2 in open-grazed community. The content ranged between 1.0 to 17.7, 0.5 to 9.7 and 0.4 to 6.6 g m^–2 for belowground and from 0.1 to 1.1, 0.1 to 0.5 and 0.1 to 0.3 g m^–2 for litter compartments in protected, semi-protected and open-grazed community respectively.A significant positive relationship was found with the nitrogen per gram dry weight in combined live and dead herbage of the study sites and the average monthly relative humidity.The distribution pattern of nitrogen in plant/soil system indicated that the major portion of nitrogen (91 per cent in the protected, 95 per cent in the semi-protected and 96 per cent in the opengrazed stands) in the system was retained in the soil while a small fraction of it (9 per cent, 5 per cent and 4 per cent on protected, semi-protected and open-grazed area respectively) resided in plant compartments. Partitioning, uptake, transfer and release of nitrogen have been discussed in detail for all three sites.     

TRIIODOTHYRONINE—Clinical Effects in Patients with Suboptimal Response to Other Thyroid Preparations

Thirty patients with evidence of hypometabolism or a clinically related condition were given triiodothyronine after suboptimal response to thyroxin or desiccated thyroid. DL- and L-isomers of triiodothyronine were used and compared. Thirteen patients (43.3 per cent) were improved and 17 (56.6 per cent) were unimproved or became worse. Side effects occurred in 9 of 34 trials (26 per cent), and could not always be eliminated by decreasing the dosage. The highest percentage of good response occurred in a small group (four of five) who received a combination of desiccated thyroid or thyroxin with supplemental triiodothyronine. Despite careful analysis of the data, no basis was found on which to predict which patients would receive benefit from the triiodothyronine. However, the occasional improvement, sometimes dramatic, suggested that a therapeutic trial with triiodothyronine in difficult or unresponsive cases of hypometabolism or hypothyroidism is justified.