A new objective histological scale for studying human photoaged skin.

BACKGROUND: A quantitative understanding of the histological alteration of the skin is important for assessing the severity of photoaging. METHODS: We performed Elastica-van Gieson staining and immunohistochemistry for decorin on 34 facial skin sections. We evaluated the alteration of collagen fibers and decorin (a modulator for collagen fibrillogenesis), according to the 5 grades of morphological change in elastic fibers that was established by Kligman (1969). The objectivity of a stage (Stages I-VI), which was established in this study, was evaluated using weighted kappa statistical analysis based on the degree of agreement in stage determination by 11 observers using a blind procedure. Correlation between the crow's-feet-area wrinkles grades of another 26 women and stages was also analyzed. RESULTS: The initial alteration of elastic fibers was observed in the deep dermis. Decorin was not detected in very severely altered skin. Based on the combination of changes in the elastic fibers, collagenic fibers, and decorin, skin tissues were categorized into 6 stages according to severity. The statistical analysis showed almost perfect agreement between observers. Significant positive correlation between stages and wrinkle scores was found. CONCLUSIONS: We propose a new objective histological scale that is useful for assessing the severity of photoaging.

Analysis of gene alterations of mitochondrial DNA D-loop regions to determine breast cancer clonality.

BACKGROUND: It has been a challenge to determine breast cancer clonality accurately. The aim of the present study was to assess methods using formalin-fixed paraffin-embedded (FFPE) tissue to differentiate new primary tumours from true recurrences that are associated with poorer prognoses and often require more aggressive treatment. METHODS: We investigated the novel method of analysing gene alterations of mitochondrial DNA D-loop region (GAMDDL) and compared it with the conventional method of analysing the X-chromosome-linked human androgen receptor (HUMARA). The FFPE sections of primary and secondary breast cancers, the non-neoplastic mammary gland, and lymph nodes were examined. RESULTS: Informative rates for HUMARA, GAMDDL, and combined analyses were 42.1%, 76.9%, and 89.5%, respectively. All of the 10 contralateral breast cancers were determined to be non-clonal. In contrast, 3 out of 8 (37.5%) of the ipsilateral secondary tumours shared a clonal origin with the primary tumour and were classified as true recurrences, whereas 4 out of 8 (50%) were classified as new primary tumours. CONCLUSION: GAMDDL analysis represents a novel and useful molecular method for examining the precise cell lineages of primary and secondary tumours, and was more accurate than HUMARA in determining clonality.

Overexpression of E2F-5 correlates with a pathological basal phenotype and a worse clinical outcome.

The purpose of the present study is to identify genes that contribute to cell proliferation or differentiation of breast cancers independent of signalling through the oestrogen receptor (ER) or human epidermal growth factor receptor 2 (HER2). An oligonucleotide microarray assayed 40 tumour samples from ER(+)/HER2(-), ER(+)/HER2(+), ER(-)/HER2(+), and ER(-)/HER2(-) breast cancer tissues. Quantitative reverse transcriptase PCR detected overexpression of a cell cycle-related transcription factor, E2F-5, in ER-negative breast cancers, and fluorescence in situ hybridisation detected gene amplification of E2F-5 in 5 out of 57 (8.8%) breast cancer samples. No point mutations were found in the DNA-binding or DNA-dimerisation domain of E2F-5. Immunohistochemically, E2F-5-positive cancers correlated with a higher Ki-67 labelling index (59.5%, P=0.001) and higher histological grades (P=0.049). E2F-5-positive cancers were found more frequently in ER(-)/progesterone receptor (PgR)(-)/HER2(-) cancer samples (51.9%, P=0.0049) and in breast cancer samples exhibiting a basal phenotype (56.0%, P=0.0012). Disease-free survival in node-negative patients with E2F-5-positive cancers was shorter than for patients with E2F-5-negative cancers. In conclusion, we identify, for the first time, a population of breast cancer cells that overexpress the cell cycle-related transcription factor, E2F-5. This E2F-5-positive breast cancer subtype was associated with an ER(-)/PgR(-)/HER2(-) status, a basal phenotype, and a worse clinical outcome.

Pathophysiological characteristics of non-alcoholic steatohepatitis-like changes in cholesterol-loaded type 2 diabetic rats.

Spontaneously Diabetic Torii (SDT) fatty rats, a new obese diabetic model, reportedly presented with features of non-alcoholic steatohepatitis (NASH) after 32 weeks of age. We tried to accelerate the onset of NASH in SDT fatty rats using dietary cholesterol loading and noticed changes in the blood choline level which is expected to be a NASH biomarker. Body weight and biochemical parameters were measured from 8 to 24 weeks of age. At 16, 20, 24 weeks, pathophysiological analysis of the livers were performed. Hepatic lipids, lipid peroxides, and the expression of mRNA related to triglyceride (TG) synthesis, inflammation, and fibrosis were evaluated at 24 weeks. Hepatic fibrosis was observed in SDT fatty rats fed cholesterol-enriched diets (SDT fatty-Cho) from 16 weeks. Furthermore, hepatic lipids and lipid peroxide were significantly higher in SDT fatty-Cho than SDT fatty rats fed normal diets at 24 weeks. Hepatic mRNA expression related to TG secretion decreased in SDT fatty-Cho, and the mRNA expression related to inflammation and fibrosis increased in SDT fatty-Cho at 24 weeks. Furthermore, SDT fatty-Cho presented with increased plasma choline, similar to human NASH. There were no significant changes in the effects of feeding a cholesterol-enriched diet in Sprague-Dawley rats. SDT fatty-Cho has the potential to become a valuable animal model for NASH associated with type 2 diabetes and obesity.

Electron microscopic identification of the intracellular secretion pathway of human G-CSF in a human tumor cell line: a comparative study with a Chinese hamster ovary cell line (IA1-7) transfected with human G-CSF cDNA.

Using monoclonal antibodies specific for human granulocyte colony-stimulating factor (G-CSF), intracellular localization of G-CSF in a G-CSF-producing human tumor cell line (CHU-2) and its ultrastructural characters were described and compared with those of a Chinese hamster ovary cell line (IA1-7) transfected with human G-CSF cDNA. The CHU-2 line, which was derived from a poorly differentiated squamous cell carcinoma of the oral cavity, preserved the character of a poorly differentiated squamous cell carcinoma. In the CHU-2 cell line, there were few cells immunohistochemically positive for G-CSF under light microscopic analysis despite the high transcription level of G-CSF cDNA and secretion of G-CSF that were comparable with cDNA-transfected IA1-7 cells. Using electron microscopy, the reaction products were localized mainly in the perinuclear space (PNS) and rough endoplasmic reticula (RER) without dilation of the cisternae, but they were very rarely found in the Golgi complex and not at all in other intracellular organelles. In contrast, most cells were positive for G-CSF in the IA1-7 cell line. Reaction products in this cell line were also demonstrated in the PNS and RER without dilation of the cisternae. These immunohistochemical findings, in conjunction with the results of Western and Northern blot analysis, suggested that G-CSF was secreted via the PNS and RER without intracellular retention.

GH and PRL gene expression by nonradioisotopic in situ hybridization in TSH-secreting pituitary adenomas.

TSH-secreting pituitary adenomas are rare. The transcriptional expression (messenger ribonucleic acids: mRNAs) of TSH beta, GH, and PRL in five patients with TSH-secreting pituitary adenoma was studied by the in situ hybridization (ISH) method in order to elucidate their multiple hormone production. These patients showed inappropriately elevated serum TSH and alpha-subunit levels as well as pituitary mass lesions. The tissues from pituitary adenomas were obtained at the time of transsphenoidal surgery and revealed immunohistochemically the expression of alpha-subunit and TSH beta in all patients. Four adenomas were immunohistochemically associated with GH or PRL localization. The presence of pituitary-specific transcriptional factor Pit-1 was demonstrated in all adenomas in the nuclei of many cells. By ISH, signals for TSH beta mRNA were present in all five cases in many adenoma cells. Expression of GH mRNA and PRL mRNA were detected not only in four adenomas in which both hormonal products were immunolocalized but also in one adenoma that was immunohistochemically negative for GH and PRL. Combined staining by ISH and immunohistochemistry revealed the expression of GH mRNA and PRL mRNA in TSH beta-immunoreactive cells. Our findings indicate that TSH-secreting adenomas are multihormone-producing and could arise from precursor or stem cells rather than from differentiated TSH-secreting cells. It is suggested that ISH combined with immunohistochemistry may provide additional detailed information concerning the multidirectional histogenesis of this rare type of adenoma.

Modulation of protein kinases and microtubule-associated proteins and changes in ultrastructure in female rat pituitary cells: effects of estrogen and bromocriptine.

This study focused on the intracellular signal transduction system and microtubule-associated proteins (MAPs), such as MAP-2 and Tau protein. The modulation of these proteins and their correlation with ultrastructural changes were investigated in rat pituitary prolactin (PRL) cells. Adult female Wistar rats were treated with estrogen and bromocriptine and their pituitary glands were removed for analysis of the expression of tubulin, MAP-2, Tau protein, protein kinase C (PKC), and calcium calmodulin (CaM) kinase. Western blot analysis showed that estrogen increased and bromocriptine decreased the expression of PKC alpha, beta 1, beta 2, CaM kinase alpha, beta, MAP-2, and Tau protein. MAP-2 and Tau protein, which are cytosolic proteins, being translated on free ribosomes, were associated with the membrane of whirling rough endoplasmic reticulum (RER) in estrogen-treated cells and dissociated with vesiculated RER induced by bromocriptine. These results suggested that the modulation of MAP-2 and Tau protein may reflect changes of PKC and CaM kinase, and that the quantitative changes and intracellular modulation of MAPs induced by estrogen and bromocriptine, i.e., estrogen-induced association and bromocriptine-induced dissociation of MAP-2 and Tau protein with membrane of RER, may reflect the dynamics of microtubules and are associated with structural changes in the RER and changes in the synthesis and intracellular transport of PRL.

Differentiation of necrotic cell death with or without lysosomal activation: application of acute liver injury models induced by carbon tetrachloride (CCL4) and dimethylnitrosamine (DMN).

We investigated the relationship between DNA degradation and lysosome activity (loss of lysosomal integrity) in necrotic cell death induced by carbon tetrachloride (CCl4) and dimethylnitrosamine (DMN): coagulation necrosis and hemorrhagic necrosis, respectively. TdT-mediated dUTP-biotin nick end-labeling (TUNEL) and enzyme histochemistry for acid phosphatase were performed in both models and results were analyzed by light microscopy, electron microscopy, and confocal laser scanning microscopy (CLSM). In the CCl(4)-injected liver, TUNEL staining was closely associated with release of lysosomal enzymes into the cytoplasm, and intranuclear deposition of lysosomal enzymes took place at an early stage of subcellular damage. In the DMN-injected liver, TUNEL-positive nuclei tended to have well-preserved lysosomes and centrally localized TUNEL signals. It was assumed that acute hepatocellular damage in the CCl4-injected liver would be characterized by necrotic cell death with lysosome activation and that damage in the DMN-injected liver would be necrotic cell death without lysosome activation. In the DMN-injected liver, DNA degradation may be selectively induced in the nuclear center, in which heterochromatin (including inactive chromatin) is believed to be a target. We concluded that necrotic cell death, i.e., DNA degradation, would be at least divided into two types, with/without association with lysosome activation, represented by necrotic cell death in the CCl4-injected liver and that in the DMN-injected liver.

Expression of plurihormonal mRNAs in somatotrophic adenomas detected using a nonisotopic in situ hybridization method: comparison with lactotrophic adenomas.

We used a nonisotopic in situ hybridization (ISH) method to investigate the expression of pituitary hormone, including glycoprotein hormone mRNAs in 17 somatotrophic and four lactotrophic adenomas. Our ISH studies of lactotrophic adenomas showed that their hormonal gene expression was confined to prolactin, whereas those of somatotrophic adenomas showed that some of them expressed plurihormonal genes. In some somatotrophic adenomas that were immunohistochemically negative for pituitary hormones, positive reactions, mainly for adrenocorticotropic hormone (ACTH), follicle-stimulating hormone beta subunit (FSH beta), and luteinizing hormone beta subunit (LH beta) mRNAs, were observed in our ISH studies. These results suggest that some somatotrophic adenomas may originate from plurihormonal primordial stem cells, which we have presumed serve as precursors for various hormone-expressing cells. It is unclear why some somatotrophic adenomas derived from plurihormonal primordial stem cells manifest clinically only as the acromegalic hyperfunction syndrome or gigantism. Additional translational factors or some other somatic mutations may play important roles in the clinical manifestations of such adenomas. In conclusion, some somatotrophic adenomas appear to be derived from plurihormonal primordial stem cells, whereas lactotrophic adenomas are well differentiated tumors that originate from lactotrophic cells, which represent the final stage of acidophilic cell line differentiation.

Electron microscopic observation of intracellular expression of mRNA and its protein product: technical review on ultrastructural in situ hybridization and its combination with immunohistochemistry.

In situ hybridization (ISH) at the electron microscopic (EM) level is essential for elucidating the intracellular distribution and role of mRNA in protein synthesis. Three different approaches have been applied by the investigators in this EM-ISH study: preembedding method; non-embedding method using ultrathin frozen sections; and postembedding method. In order to obtain satisfactory morphological preservation and retain the messages, we routinely utilized 6 microns-thick frozen sections fixed in 4% paraformaldehyde for the preembedding method and tissues embedded in LR White resin for the postembedding method. The hybridization signal intensity by the postembedding method was lower, and non-specific signals were relatively frequent, in comparison with the preembedding method. The preembedding method thus appears to be easier and better than the postembedding method from the viewpoint of applicability and preservation of mRNA, although quantitative analysis of the expression of mRNA is rather difficult in the preembedding method. EM-ISH is considered to be an important tool for clarifying the intracellular localization of mRNA and the exact site of specific hormone synthesis on the rough endoplasmic reticulum. The simultaneous visualization of mRNA and encoded protein in the same cells using preembedding EM-ISH and subsequent postembedding immunoreaction with protein A colloidal gold complex is also described. This ultrastructural double-staining method for mRNA and encoded protein can be expected to provide an important clue for elucidating the intracellular correlation of mRNA translation and secretion of translated protein.

In situ nick end-labeling detects necrosis of hippocampal pyramidal cells induced by kainic acid.

It is believed that in situ nick end-labeling (ISNEL) is an easy and selective method for detecting apoptosis in situ. To test whether ISNEL selectively detects apoptosis but not necrosis, we investigated the kainic acid (KA)-induced neuronal death with ISNEL, comparing with the results of gel electrophoresis and electron microscopy. Many degenerating neurons (ca. 50%) in the hippocampal CA1 area and amygdaloid complex were intensely stained with ISNEL 1-3 days after intraperitoneal injection of KA. Although most of the ISNEL-positive neurons displayed a pathological feature of necrosis, a small number of them displayed apoptosis-like changes when examined by electron microscopic observation. It should be noteworthy that ISNEL recognizes at least a certain form of necrosis and is not selective for apoptosis.

Phospholipase C-dependent hydrolysis of phosphatidylcholine hydroperoxides to diacylglycerol hydroperoxides and its reduction by phospholipid hydroperoxide glutathione peroxidase.

We have shown that 1,2-diacylglycerol hydroperoxides activate protein kinase C (PKC) as efficiently as does phorbol ester [Takekoshi S, Kambayashi Y, Nagata H, Takagi T, Yamamoto Y, Watanabe K. Activation of protein kinase C by oxidized diacylglycerol. Biochem Biophys Res Commun 1995; 217: 654-660]. 1,2-Diacylglycerol hydroperoxides also stimulate human neutrophils to release superoxide whereas their hydroxides do not [Yamamoto Y, Kambayashi Y, Ito T, Watanabe K, Nakano M. 1,2-Diacylglycerol hydroperoxides induce the generation and release of superoxide anion from human polymorphonuclear leukocytes. FEBS Lett 1997; 412: 461-464]. One of the proposed mechanisms for the formation of 1,2-diacylglycerol hydroperoxides is the hydrolysis of phosphatidylcholine hydroperoxides by phospholipase C (PLC). To confirm this hypothesis, we incubated 1-palmitoyl-2-linoleoyl-phosphatidylcholine (PLPC) liposomes containing PLPC hydroperoxides (PLPC-OOH) with Bacillus cereus PLC and found 1-palmitoyl-2-linoleoylglycerol (PLG) and its hydroperoxide (PLG-OOH) were produced. PLC hydrolyzed the two substrates without preference, as the yields of PLG and PLG-OOH were the same even though cholesterol was incorporated into liposomes to increase bilayer integrity. Phospholipid hydroperoxide glutathione peroxidase (PHGPX) reduced PLG-OOH to its hydroxide in the presence of glutathione while the conventional cytosolic glutathione peroxidase did not. These data suggest that PLC hydrolyzes oxidized biomembranes to give 1,2-diacylglycerol hydroperoxides for PKC stimulation but PHGPX may prevent neutrophil stimulation by reducing 1,2-diacylglycerol hydroperoxides to their hydroxides.

Biochemical and immunohistochemical analysis of orotic acid-induced fatty liver.

Orotic acid-induced fatty livers were examined by biochemical and immunohistochemical approaches. Lipid peroxide levels by the thiobarbituric acid method and glutathione-peroxidase (GSH-PO) activity in the liver homogenates from orotic administered rats were similar to those of controls. Immunohistochemical localization of GSH-PO in orotic acid-induced fatty liver was mainly observed in the portal zone of the hepatic lobules. This staining pattern of GSH-PO was similar to that of the controls. No remarkable changes in GSH-PO staining patterns were detected in orotic acid-induced fatty liver. Our data strongly suggested that no lipid peroxidation is actively involved in the genesis of fatty liver due to the administration of orotic acid, and GSH-PO a protective enzyme against lipid peroxidation, was not inhibited by orotic acid-induced fatty liver.

Suppression of messenger ribonucleic acid for glutathione peroxidase in chemically induced rat hepatocellular carcinoma and its biological significance.

Deviations of the enzyme activity, immunoreactivity and messenger ribonucleic acid (mRNA) levels of glutathione peroxidase (GSH-PO) in 3'-methyl-4-dimethylaminoazobenzene- induced hepatocellular carcinoma of the rat were investigated. Enzyme activities of GSH-PO were significantly lower in hepatocellular carcinomas than those in the normal control rat liver. Immunohistochemically, GSH-PO was strongly localized in normal hepatocytes, but was only faintly stained in hepatocellular carcinoma cells. Heterogeneous staining patterns of GSH-PO were observed among individual cancer cells. In Northern blot analysis, GSH-PO mRNA in the cancer tissue was decreased to two thirds of the level in normal hepatocytes. It was suggested that suppressed expression of GSH-PO in carcinogen-induced hepatocellular carcinomas occurred at the level of mRNA transcription.

Immunolocalization of glutathione-peroxidase (GSH-PO) in the rat ventral prostate: effects of castration and administration of testosterone.

Immunolocalization of glutathione-peroxidase (GSH-PO) in the rat ventral prostate was studied in the presence and absence of androgen. Male Sprague-Dawley rats were divided into four experimental groups. Group 1 consisted of intact controls. In group 2, rats were sacrificed two days after castration. In groups 3 and 4, rats were injected subcutaneously with 1 mg of testosterone-propionate daily, for three or seven days, beginning two days after castration. The intensity of GSH-PO staining in the glandular epithelial cells of the ventral prostate decreased after castration, but recovered following treatment with testosterone. Furthermore, the prostatic GSH-PO mRNA levels were diminished in the castrated rat ventral prostate but greatly increased by testosterone. These findings strongly suggest that the expression of GSH-PO in the glandular epithelial cells of the rat ventral prostate is dependent on testosterone.

Antioxidative action of flavonoids, quercetin and catechin, mediated by the activation of glutathione peroxidase.

Antioxidative action of flavonoids have been attracted attention of many investigators and a good deal of studies on it were reported. While their interests were mostly centered to the direct scavenging action of flavonoids against free radicals and active oxygen species, we expected that the interaction of flavonoids and intracellularly occurring antioxidative agents such as glutathione peroxidase (GSH-PO) could synergistically enhance their antioxidative activities. For this purpose, cultured rat hepatocytes (BL-9), which are highly expressing GSH-PO, were employed. One group of the cells were cultured with Se deficient media (Se(-) cells) to diminish the activity and the expression of GSH-PO protein and mRNA, and the other group was cultured with Se supplemented media (Se(+) cells). The oxidative cell damage was induced by the addition of H2O2 and two representative antioxidative flavonoids, quercetin and catechin, were added to the media to test their cytoprotective action. In Se(+) cells, the remarkable cytoprotective activity of those flavonoids were confirmed, whereas none of such activity was evidenced in Se(-) cells. It was proved that the intracellular antioxidative function of flavonoids requires the interaction with GSH-PO, at least in the cells expressing the enzyme. Interestingly, the flavonoid activated GSH-PO clearly, and its mechanism is discussed.

Expression of glutathione-peroxidase (GSH-PO) in the rat ventral prostate--effect of castration and administration of testosterone.

Immunolocalization of glutathione-peroxidase (GSH-PO), apoptosis and bcl-2 protein in the rat ventral prostate was investigated in the presence or absence of androgen. Male Sprague-Dawley rats were divided into four experimental groups. Group 1 consisted of intact controls. In group 2, rats were sacrificed two days after castration. In groups 3 and 4, rats were administered subcutaneously 1 mg/animal of testosterone-propionate daily for three or seven days at two days after castration. The intensity of GSH-PO staining in the glandular epithelial cells of the ventral prostate was remarkably decreased after castration (Group 2), and it clearly recovered when testosterone was administered (Groups 3 and 4) to the castrated rats. The prostatic GSH-PO mRNA levels were diminished in the castrated rat ventral prostate but greatly increased by testosterone (Groups 3 and 4). Furthermore, castration (Group 2) induced apoptosis in the prostatic glandular epithelial calls and the apoptosis was reduced by testosterone-administration (Groups 3 and 4) to the castrated rats. In groups 3 and 4, expression of bcl-2 protein was clearly detected in the glandular epithelial cells of the ventral prostate. These findings strongly suggested that expression of GSH-PO and bcl-2 protein in the glandular epithelial cells of the rat ventral prostate is testosterone-dependent.

Ki-67 and minichromosome maintenance-7 (MCM7) expression in canine pituitary corticotroph adenomas.

Pituitary-dependent hyperadrenocorticism (PDH) caused by pituitary corticotroph adenoma is a common endocrine disorder in dogs. The ratio between pituitary height and the area of the brain (P/B) has been used to evaluate the pituitary size. A P/B ratio > 0.31 indicates an enlarged pituitary, whereas a P/B ratio ≤ 0.31 indicates a nonenlarged pituitary. The aim of this study was to investigate the expression of proliferation markers Ki-67 and minichromosome maintenance-7 (MCM7) in canine corticotroph adenomas in enlarged and in nonenlarged pituitaries and to evaluate their relation with the size of canine pituitary corticotroph adenomas. Ki-67 and MCM7 expression in ACTH-positive tumor cells was determined by dual-labeling immunohistochemistry in resected corticotroph adenomas from 15 dogs with PDH. The mean ± SD Ki-67 labeling index (LI) was 0.55% ± 0.59% in corticotroph adenomas with nonenlarged pituitaries and 1.6% ± 0.6% in adenomas with enlarged pituitaries. The MCM7 LI in corticotroph adenomas with nonenlarged pituitaries and in adenomas with enlarged pituitaries was 2.9% ± 2.2% and 10.9% ± 3.7%, respectively. The Ki-67 LI and MCM7 LI were significantly greater in the adenomas with enlarged pituitaries than in the adenomas with nonenlarged pituitaries (P < 0.01 and P < 0.01, respectively). The MCM7 LI was significantly greater than the Ki-67 LI in adenomas (P < 0.01). The Ki-67 LI was positively correlated with the MCM7 LI (r = 0.820, P < 0.01), and the P/B ratio was positively correlated with the Ki-67 LI (r = 0.560, P = 0.03) and the MCM7 LI (r = 0.854, P < 0.01). In conclusion, canine corticotroph adenomas in enlarged pituitaries show greater proliferation potential than do adenomas in nonenlarged pituitaries. MCM7 expression was significantly greater than Ki-67 expression in canine pituitary corticotroph adenomas. Thus, MCM7 may be superior to Ki-67 as a proliferation marker in pituitary tumors.

Loss of heterozygosity alterations associated with progesterone therapy in endometrial hyperplasia and adenocarcinoma.

Loss of heterozygosity (LOH) was analyzed in four patients with endometrial hyperplasia (EH) with atypia (two patients) and without atypia (two patients) and in five patients with endometrial adenocarcinoma (EAC) to clarify the clinicopathologic relationship between genetic alterations and hormone therapy. Each patient was initially administered high-dose medroxyprogesterone acetate (MPA) as a uterine-sparing treatment. The five microsatellite markers used to analyze LOH were at chromosomal loci 8p22.1, 8p21, 8p21.3, 8p22, and 8p22. DNA was extracted from paraffin-embedded sections before, during, and after MPA therapy using laser capture microdissection. As a result, LOH was more frequently detected after MPA therapy (overall ratios were 16, 17, and 29% before, during, and after MPA therapy, respectively). LOH is more easily detected in EH loci than in EAC loci before MPA. For EAC, initial LOH detection on chromosome 8 may be related to an incomplete response to MPA, but negative LOH does not guarantee a favorable treatment outcome. For EH or atypical endometrial hyperplasia, it is unknown whether LOH alteration associated with MPA therapy is related to atypia of the disease.

Effects of ipriflavone on calcitonin synthesis in C cells of the rat thyroid.

Ipriflavone is known to stimulate calcitonin (CT) secretion from the thyroid glands of female animals, but the exact mechanism of this action remains unknown. In the present study, an increase of CT production in thyroid C cells of female rats, but not of male rats, was proven immunohistochemically. Furthermore, the parallel increase of CT mRNA in those thyroid glands was confirmed by Northern blot analysis. These results proved that ipriflavone stimulates not only CT secretion but also CT synthesis in thyroid C cells and that the changes were gender dependent (greater in females). In order to investigate the effect of estrogen on the ipriflavone-induced increase of CT in female rat thyroid gland, CT and CT mRNA in the thyroid glands of untreated, ovariectomized, and estrone-treated (postovariectomy) rats were examined by both immunohistochemistry and Northern blot technique. Serum levels of CT and calcium were also examined. Against expectation, estrone failed to produce any significant effect on the ability of ipriflavone to induce CT synthesis and secretion.