Specific progesterone receptors in dimethylbenzanthracene (DMBA)-induced mammary tumors.

Properties of a progesterone receptor present in the cytosol (105,000 xg supernatant) of dimethylbenzanthracene (DMBA)-induced mammary tumors were studied using the highly potent progestin [3H]R 5020 (17, 21-dimethyl-19-nor-pregna-4, 9-diene-3,20-dione). As shown by sucrose gradient analysis, specific binding of [3H] R 5020 is associated with components migrating at 7-8S and 4S. Low affinity binding of the synthetic progestin is eliminated by treatment with dextran-coated charcoal. [3H] R 5020 binding is highly progestin-specific since it is easily displaced by unlabelled norgestrel, R 5020 and progesterone while estradiol-17beta, dihydrotestosterone, testosterone, testosterone and diethylstilbestrol have much lower activity. Dexamethasone and cortisol have little, if any, effect on [3H] R 5020 binding.     

Differential oxidation of deoxyribose in DNA by gamma and alpha-particle radiation

Emerging evidence points to the importance of deoxyribose oxidation in the toxicity of oxidative DNA damage, including the formation of protein-DNA crosslinks and base adducts. With the goal of understanding the differences in deoxyribose oxidation chemistry known to occur with different oxidants, we have compared the formation of one product of 3'-oxidation of deoxyribose in DNA, 3'-phosphoglycolaldehyde (PGA) residues, in isolated DNA and cells exposed to ionizing radiations. A recently developed gas chromatography/negative chemical ionization mass spectrometry method was used to quantify PGA residues in purified DNA and in human TK6 lymphoblastoid cells exposed to gamma radiation (60Co) and alpha particles (241Am). The level of PGA residues was then correlated with the total quantity of deoxyribose oxidation determined by plasmid topoisomer analysis. Alpha-particle irradiation (0-100 Gy) of purified DNA in 50 mM potassium phosphate (pH 7.4) produced a linear dose response of 0.13 PGA residues per 10(6) nucleotides per gray. When normalized to an estimate of the total number of deoxyribose oxidation events (2.0 per 10(6) nucleotides per gray), PGA formation occurred in 7% (+/-0.5) of deoxyribose oxidation events produced by alpha-particle radiation. In contrast, the efficiency of PGA formation in gamma-irradiated DNA was found to be 1% (+/-0.02), which indicates a shift in the chemistry of deoxyribose oxidation, possibly as a result of the different track structures of the two types of ionizing radiation. Studies with gamma radiation were extended to TK6 cells, in which it was observed that gamma radiation produced a linear dose response of 0.0019 PGA residues per 10(6) nucleotides per gray. This is consistent with an approximately 1000-fold quenching effect in cells, similar to the results of other published studies of oxidative DNA damage in vivo.     

Repopulation kinetics of rat rhabdomyosarcoma tumors following single and fractionated doses of low-LET and high-LET radiation

Measurements were made of clonogenic cell survival in rat rhabdomyosarcoma tumors as a function of time following in situ irradiation with single or fractionated doses of 225-kVp X rays or with 557-MeV/u neon ions in the distal position of a 4-cm extended-peak ionization region. Single doses of 20 Gy of X rays or 7 Gy of peak neon ions reduced the initial surviving fraction to approximately 0.025 for each modality. Daily fractionated doses (four fractions in 3 days) of either peak neon ions (1.75 Gy per fraction) or X rays (6 Gy per fraction) achieved a cell survival of approximately 0.02-0.03 after the fourth dose of radiation. In the single-dose experiments, significant 5- and 10-fold decreases in the fraction of clonogenic cells were observed between the third and fourth days after irradiation with peak neon ions and X rays, respectively. After the sixth day postirradiation, the residual clonogenic cells exhibited a rapid burst of proliferation leading to doubling times for the surviving cell fractions of approximately 1.5 days. Radiation-induced growth delay was consistent with the cellular repopulation dynamics. In the fractionated-dose experiments with both radiation modalities, a large delayed decrease in cell survival was observed at 1-3 days after completion of the fractionated-dose schedule. Cellular repopulation was consistent with postirradiation tumor volume regression and regrowth for both radiation modalities. The extent of decrease in survival following the four-fraction radiation schedule was approximately two times greater in X-irradiated than in neon-ion-irradiated tumors that produced the same survival level immediately after the fourth dose. Mechanisms underlying the marked reduction in cell survival 3-4 days postirradiation are discussed, including the possible role of a toxic host cell response against the irradiated tumor cells.     

Activation of c-myc and c-K-ras oncogenes in primary rat tumors induced by ionizing radiation.

An activated K-ras oncogene was detected by transfection in NIH 3T3 cells and by Southern blot analysis in 6 of 12 rat skin tumors induced by ionizing radiation. The DNA from 10 of the 12 tumors also showed c-myc gene amplification and restriction polymorphisms. Evidence for tissue specificity was observed in patterns of oncogene activation, with each of three clear cell carcinomas exhibiting activation of both c-myc and K-ras oncogenes.     

DMBA induced DNA damage and repair in mammary epithelial cells in vitro measured by a nick translation assay

A new E. coli DNA polymerase I directed nick translation assay was used for measuring 7,12-dimethylbenz[a]anthracene-induced in situ DNA damage and repair in mouse mammary epithelial cells in monolayer culture. The nick translation assay was capable of detecting a DMBA-dose dependent significant increase of DNA damage, and the same assay also allowed monitoring of the DNA repair activity provoked by DMBA treatment of the epithelial cells. This relatively simple method thus provides a rapid assay for carcinogen-induced in situ DNA damage and repair in an epithelial cell tumorigenic system.     

Persistent changes in gene expression induced by estrogen and progesterone in the rat mammary gland.

Epidemiological studies have consistently shown that an early full-term pregnancy is protective against breast cancer. We hypothesize that the hormonal milieu that is present during pregnancy results in persistent changes in the pattern of gene expression in the mammary gland, leading to permanent changes in cell fate that determine the subsequent proliferative response of the gland. To investigate this hypothesis, we have used suppression subtractive hybridization to identify genes that are persistently up-regulated in the glands of E- and progesterone (P)-treated Wistar-Furth rats 28 d after steroid hormone treatment compared with age-matched virgins. Using this approach, a number of genes displaying persistent altered expression in response to previous treatment with E and P were identified. Two markers have been characterized in greater detail: RbAp46 and a novel gene that specifies a noncoding RNA (designated G.B7). Both were persistently up-regulated in the lobules of the regressed gland and required previous treatment with both E and P for maximal persistent expression. RbAp46 has been implicated in a number of complexes involving chromatin remodeling, suggesting a mechanism whereby epigenetic factors responsible for persistent changes in gene expression may be related to the determination of cell fate. These results provide the first support at the molecular level for the hypothesis that hormone-induced persistent changes in gene expression are present in the involuted mammary gland.     

Identification of genes responsive to gamma radiation in rat hepatocytes and rat liver by cDNA array gene expression analysis

The mechanisms underlying hepatocellular damage after irradiation are obscure. We identified genes induced by radiation in isolated rat hepatocytes in vitro by cDNA array gene expression analysis and then screened in vivo experiments with those same genes using real-time PCR and Western blotting. Hepatocytes were irradiated and cDNA array analyses were performed 6 h after irradiation. The mRNA of differentially expressed genes was quantitatively analyzed by real-time PCR. cDNA array analyses showed an up-regulation of 10 genes in hepatocytes 6 h after irradiation; this was confirmed by real-time PCR. In vivo, rat livers were irradiated selectively. Treated and sham-irradiated controls were killed humanely 1, 3, 6, 12, 24 and 48 h after irradiation. Liver RNA was analyzed by real-time PCR; expression of in vivo altered genes was also analyzed at the protein level by Western blotting. Up-regulation was confirmed for three of the in vitro altered genes (multidrug resistance protein, proteasome component C3, eukaryotic translation initiation factor 2). Histologically, livers from irradiated animals were characterized by steatosis of hepatocytes. Thus we identified genes that may be involved in liver steatosis after irradiation. The methods shown in this work should help to further clarify the consequences of radiation exposure in the liver.     

Phospholipase C activity in normal rat mammary tissues and in DMBA-induced rat mammary tumors

Employing phosphatidylinositol as the substrate, phospholipase C (PLC) activity was measured in various cellular fractions derived from DMBA-induced rat mammary tumors and mammary tissues from 12-14 day pregnant rats. In the 2,000g pellet, 10,000g pellet, 100,000g pellet and 100,000g supernatant fractions, PLC activity was more than 5 fold higher in the fractions derived from the neoplastic tissues. This was true whether PLC activity was expressed on the basis of protein content or 5' nucleotidase activities.     

Enhancement of DMBA induced mammary tumours by intermittent whole body hyperthermia (42 degrees C)

Wistar Female rats bearing DMBA induced mammary tumours were subjected to whole body hyperthermia 42 degrees C dry heat exposure for 15 minutes daily for 6 weeks. The control group was maintained at a room temperature of 25 degrees C. Hyperthermia induced significant growth stimulation of breast tumour compared to the controls. Plasma estradiol was slightly decreased while total T4 and TSH values remained unchanged in heat stressed rats. Plasma prolactin was significantly increased together with enhanced synthetic activity of pituitary prolactin cells. It is concluded that heat acting as stressor accelerates breast tumor growth, probably by influencing synthesis of prolactin. Therefore the hormone dependency of tumours should be considered before hyperthermia is used as an anticancer modality.     

Chlorophyll mutations induced by gamma radiation in Phaseolus vulgaris L]

In a study of chlorophyll mutants of Phaseolus vulgaris L. through Co60 gamma radiation, five types of mutants, classified as albino, cream, yellow, yellow-green and light green were obtained; all were lethal; their segregation was always proportionally lower than the Mendelian. Gamma radiation-induced mutations in black beans do not depart significantly from those obtained elsewhere in barley and wheat.     

Resistance to schistosome infection in Biomphalaria glabrata induced by gamma radiation

Two strains of Biomphalaria glabrata were studied with respect to the effects of ionizing radiation on their susceptibility to Schistosoma mansoni infection. Gamma radiation at levels of 3.5 and 5 krad did not induce susceptibility in the resistant S-3 strain, but was found to initiate resistance in the susceptible PR-1 strain. In an attempt to understand the induced resistance in irradiated snails, histopathologic examinations and analyses of snail hemolymph were performed. Results indicated that miracidia invading irradiated snails were quickly surrounded and encapsulated by amoebocytes. Similarly, alterations in the hemolymph of irradiated snails suggested that radiation induced aging. It is suggested that radiation-altered snails may be of value in studying the defense mechanisms of these organisms.     

Repair of DNA damage induced by gamma radiation and UV and gamma mimetics in virus-infected human cells

The method of chromatography of cell lysates on the columns with hydroxyapatite (HAP) and the method of ultracentrifugation of cell lysates in neutral sucrose gradient were used to study the mutagen-induced repair activity of human cells HEp-2 noninfected and chronically infected with measles and rubella viruses in order to determine the sedimentation properties of complexes containing DNA. Gamma-radiation, bleomycin, 4-nitroquinoline-1-oxide, and mitomycin C were used as DNA damaging agents. It was shown that the chronic infectious process inhibited repair of DNA damages induced by 4-nitroquinoline-1-oxide and mitomycin C and did not influence repair of DNA lesions caused by gamma-radiation and bleomycin.     

Differential proteome and phosphoproteome signatures in human T‐lymphoblast cells induced by sirolimus

Objectives: The present study was designed to investigate early proteome and phosphoproteome changes during inhibition of lymphocyte proliferation induced by sirolimus (SRL). Materials and methods: Proliferation assays were conducted using human CCRF‐CEM T lymphoblasts under different SRL concentrations. Total protein lysates after SRL treatment were used to identify significantly regulated proteins and phosphorylated proteins by 2‐DE and Q‐TOF Ultima Global mass spectrometer. Results and conclusions: Incubation with 2.5 μmol/l SRL resulted in a ～ 70% inhibition of cell proliferation. Cells incubated with 2.5 μmol/l for 30 min showed a differential phosphorylation pattern with one higher (TCPQ) and six lower phosphorylation signals (TBA1B, VIME, HNRPD, ENPL, SEPT9, PLSL). On investigating the differential protein expression, five proteins were found to be up‐regulated (ECHB, PSB3, MTDC, LDHB and NDKA) and four were down‐regulated (EHD1, AATC, LMNB1 and MDHC). Nine of these differentially regulated proteins/phosphoproteins (TCPQ, TBA1B, VIME, HNRPD, ENPL, ECHB, PSB3, LDHB and LMNB1) showed significant interaction potential, through binding protein YWHAZ using MINT software. Conclusions: We report for the first time the simultaneous early influence of SRL on phosphorylation status and on protein expression in the total proteome of CCRF‐CEM T lymphoblasts and predict that 56% of the proteins interact with each other, highlighting significance of these results.     

Cytochemical study of the RNA in rat adenohypophysis cells during mammary carcinogenesis induced by 7-12-dimethyl-(a)-benzanthracene (DMBA)

A cytochemical study of RNA was carried out in gonadotropic basophils and oxyphils of adenohypophysis of albino nonstrain rats during DMBA-induced mammary cancer growth. A stage character is found for changes in the RNA content depending on the mammary carcinogenesis phase. This shows that DMBA carcinogenic action besides a direct damage effect on the mammary gland is realized to a considerable extent through the endocrine system.     

Estrogen regulation of superoxide dismutase in normal rat mammary tissues and mammary tumors

Multiple electrophoretic molecular variants of superoxide dismutase were demonstrated in normal rat mammary tissues and DMBA-induced rat mammary tumors. The specific activities of $\text{Cu}\text{Zu}$ superoxide dismutase in mammary tumors of estrogen-treated rats were not significantly different from those activities seen in normal rat mammary tissues. However, the enzyme activities of mammary tumors from untreated rats (no estrogen) were significantly lower than the activities of normal rat mammary tissues. Exogenous estrogen appeared to raise superoxide dismutase levels in DMBA-induced rat mammary tumors to those levels seen in normal rat mammary tissues.     

Phosphoinositide phosphorylation precedes growth in rat mammary tumors

DMBA-induced rat mammary tumors were used to study the possible association of phosphoinositide phosphorylation to tumor growth. These membranous enzymatic activities were measured during various stages of tumor growth induced by pharmacological manipulation of plasma prolactin level. An increase in phosphorylation of both phosphatidyl inositol and phosphatidyl inositol 4-phosphate preceded the growth induced by prolactin concomitantly with an increase in tyrosine phosphorylation. Good correlation (r = 0.87) existed between the tyrosine kinase activity and phosphatidyl inositol kinase activity of 21 individual tumors taken from animals at different stages of hormonal manipulation. Phosphoinositide phosphorylation was inhibited by quercetin and was not affected by cAMP, similar to tyrosine kinase. Phosphorylation of angiotensin II by tyrosine kinase was inhibited by 0.2 mg/ml phosphatidyl inositol 4 phosphate or phosphatidyl inositol 4,5-bisphosphate.     

Hormonal dependency of experimental mammary tumors induced by an estro-progestational combination

Mammary tumours were induced in rats by administration of an estradiol-progesterone association. These tumours offered close analogies with human mammary tumours. The estradiol-receptor was found both in cytosol and nuclei and was more abundant in nuclei than in cytosol. The major part of its binding sites was occupied by endogenous hormone. On the other hand, a high level of progesterone receptor was present in tumoral cytosol. This fact gave evidence of the complete activity of the estradiol receptor. The presence of estradiol and progesterone receptors in mammary tumors induced by these hormones substantiate their hormone-dependence.