HDAC Inhibition Induces CD26 Expression on Multiple Myeloma Cells via the c-Myc/Sp1-mediated Promoter Activation.

CD26 is ubiquitously and intensely expressed in osteoclasts in patients with multiple myeloma, whereas its expression in plasma cells of patients with multiple myeloma is heterogeneous because of its cellular diversity, immune escape, and disease progression. Decreased expression levels of CD26 in myeloma cells constitute one of the mechanisms underlying resistance to humanized anti-CD26 mAb therapy in multiple myeloma. In the current study, we show that histone deacetylase inhibition (HDACi) with broad or class-specific inhibitors involves the induction of CD26 expression on CD26neg myeloma cells both transcriptionally and translationally. Furthermore, dipeptidyl peptidase Ⅳ (DPPⅣ) enzymatic activity was concomitantly enhanced in myeloma cells. Combined treatment with HDACi plus CD26mAb synergistically facilitated lysis of CD26neg myeloma cells not only by antibody-dependent cellular cytotoxicity but also by the direct effects of mAb. Of note, its combination readily augmented lysis of CD26neg cell populations, refractory to CD26mAb or HDACi alone. Chromatin immunoprecipitation assay revealed that HDACi increased acetylation of histone 3 lysine 27 at the CD26 promoter of myeloma cells. Moreover, in the absence of HDACi, c-Myc was attached to the CD26 promoter via Sp1 on the proximal G-C box of myeloma cells, whereas, in the presence of HDACi, c-Myc was detached from Sp1 with increased acetylation of c-Myc on the promoter, leading to activation of the CD26 promoter and initiation of transcription in myeloma cells. Collectively, these results confirm that HDACi plays crucial roles not only through its anti-myeloma activity but by sensitizing CD26neg myeloma cells to CD26mAb via c-Myc/Sp1-mediated CD26 induction, thereby augmenting its cytotoxicity. SIGNIFICANCE: There is a desire to induce and sustain CD26 expression on multiple myeloma cells to elicit superior anti-myeloma response by humanized anti-CD26 mAb therapy. HDACi upregulates the expression levels of CD26 on myeloma cells via the increased acetylation of c-MycK323 on the CD26 promoter, leading to initiation of CD26 transcription, thereby synergistically augments the efficacy of CD26mAb against CD26neg myeloma cells.

The effect of aromatase inhibitor on controlled ovarian stimulation for oocyte cryopreservation in adolescent and young cancer patients.

AIM: To determine the impact of aromatase inhibitor (AI) use in oocyte cryopreservation among Japanese adolescent and young adult (AYA) cancer patients for fertility preservation, we evaluated the oocyte cryopreservation outcomes following AI therapy in combination with the follicular phase start (FPS) and random start (RS) protocols. METHODS: This retrospective study included 81 cycles of controlled ovarian stimulation (COS) among 73 AYA patients with cancer who underwent oocyte cryopreservation to maintain fertility. The outcome measures were the total number of matured oocytes that were retrieved and cryopreserved, as well as their maturation rates. The AI (+) and AI (-) groups were compared using the RS and FPS protocols. RESULTS: Our results showed that the combined use of AI and COS decreases serum E2 levels and maintains the number of retrieved and cryopreserved mature oocytes. We also confirmed the efficacy of the RS protocol, which was found to have comparable outcomes to that of the FPS protocol in both AI (+) and AI (-) groups. CONCLUSION: The combined use of AI and COS is beneficial for oocyte cryopreservation in patients with estrogen-sensitive cancer, regardless of the menstrual cycle phase of COS initiation.

Primary malignant melanoma of uterine cervix treated with pembrolizumab as adjuvant immunotherapy.

This is the case report of primary malignant melanoma (MM) of uterine cervix treated by immune checkpoint inhibitor: the Pembrolizumab. Despite the merge of the novel drugs that has been strikingly improving prognosis of MM, we still struggle treatment of MM of uterine cervix that has aggressive characteristics with unknown etiology. We present our case to contribute its rarity of the disease case report, the primary MM of the uterine cervix that had poor response to pembrolizumab and had OS of 6 months. The treatment ineffectiveness is mainly considered for mucosal MM of low tumor mutation burden and its unusual type of pathology. Accumulation of retrospective studies exclusively on cervical melanoma needs to be proceeded to investigate on characteristics between poor and long survival to establish standardized treatment.

Examining the Beneficial Aspects of Nutritional Guidance Using Estimated Daily Salt Intake in Cancer Patients with Ischemic Heart Disease.

BACKGROUND The outcomes associated with nutritional guidance for patients with ischemic heart disease undergoing cancer treatment have not been explored. We examined the effects of nutritional guidance using estimated daily salt intake in cancer patients with ischemic heart disease. MATERIAL AND METHODS We examined the data from physical examinations and laboratory assessments of 27 patients with suspected excessive salt intake who underwent coronary angiography for the first time and received nutritional guidance on their next visit to the Department of Cardiology of Shizuoka Cancer Center between May 2018 and March 2020. Salinity measurement was not used in the nutritional guidance method, but the patients were instructed to reduce consumption of salt-containing foods. We compared the frequency of the estimated daily salt intake with the frequency of categories requiring salt control (food, cooking, and table salts). RESULTS The median age of the participants was 74 (range, 63-86) years. The estimated daily salt intake and the rate of change in the triglyceride level were negatively correlated (r=-0.61, P<0.01). The estimated daily salt intake was reduced in 16 cases; there was a relative decrease in the frequency of food intake among categories requiring salt control compared with that in the nonimproved cases (P<0.01). No difference was found between the cancer stage and the affected site of the digestive system in either group (P=0.64, P=0.39). CONCLUSIONS Nutritional guidance on dietary habits without using salinity measurement was beneficial in preventing ischemic heart disease and food intake reduction in cancer patients.

Energy metabolism profile of the effects of amino acid treatment on hepatocytes: Phenylalanine and phenylpyruvate inhibit glycolysis of hepatocytes.

OBJECTIVES: Amino acids are not only the building blocks of proteins, but also can be metabolized to energy substances or function as signaling molecules. The aim of this study was to profile whether amino acid treatment (essential amino acids and alanine) affects the energy metabolism (glycolysis, mitochondrial respiration) of cultured hepatocytes. METHODS: AML12 hepatocytes were treated with 5 mM of each amino acid for 1 h and the energy metabolism was then measured by using an extracellular flux analyzer. RESULTS: The results showed that phenylalanine and lysine decreased the extracellular acidification rate (ECAR), an indirect indicator of glycolysis, whereas isoleucine and histidine increased the ECAR. Amino acids did not affect the oxygen consumption rate, an indirect indicator of mitochondrial respiration. The glycolysis stress test revealed that treatment of the hepatocytes with phenylalanine inhibited glycolysis when the concentration of the substrate for glycolysis was sufficient in cultured media. We also investigated the effect of metabolites derived from conversion of phenylalanine on glycolysis in hepatocytes and found that phenylpyruvate inhibited glycolysis, whereas tyrosine and phenylethylamine did not affect glycolysis. CONCLUSIONS: The findings from the present study complement basic knowledge of the effects of amino acid treatment on energy metabolism in cultured hepatocytes and indicate that phenylalanine and phenylpyruvate inhibit glycolysis.

The association of urinary estrogen levels with urinary isoflavone levels: Difference between premenopausal women and postmenopausal women.

Results of studies on the associations of soy food intake with urinary estrogen levels in premenopausal women and in postmenopausal women have been inconsistent. We examined the associations of urinary isoflavone levels as well as soy food intake with estrone (E1) and estradiol (E2) in pre- and postmenopausal women. In addition, we compared the levels of isoflavones, E1 and E2 across current hormone users such as those receiving hormone replacement therapy and those using oral contraceptives and non-users among both pre- and postmenopausal women. Urinary levels of isoflavones, E1 and E2 in 498 women (36 hormone users and 462 non-users) were analyzed. Premenopausal women with a higher frequency of soy food intake had higher urinary isoflavone levels, but there were no significant associations between E1 and E2 levels and urinary isoflavone levels. Levels of E1 and E2 in hormone users were significantly lower than those in hormone non-users among premenopausal women, but levels of E1 and E2 in hormone users were significantly higher than those in hormone non-users among postmenopausal women. Postmenopausal women with a higher frequency of soy food intake had higher urinary isoflavone levels, and postmenopausal women with high urinary isoflavone levels had significantly higher E1 and E2 levels. In conclusion, the associations of urinary isoflavone levels with urinary estrogen levels differed with menopausal status. Urinary levels of E1 and E2 were high in postmenopausal women with high urinary isoflavone levels but not in premenopausal women with high urinary isoflavone levels.

[Leanness, obesity, and breast cancer risk-different impact of body weight on breast cancer risk according to women's life stages].

Numerous epidemiological studies, although not all, in Western countries have reported a possible differential impact of BMI on breast cancer risk in women of various lifestages. Among premenopausal women, a number of epidemiological studies in Western populations suggested a weak inverse association between BMI and breast cancer risk. Conversely, there exists substantial evidence for a statistically significant positive association between body weight and breast cancer risk among postmenopausal women. The cumulative exposure to estrogen throughout a woman's life is one of the significant risk factors for breast cancer. After menopause, adipose tissue is a major source of estrogen. Therefore, an increase in body fat after menopause is one of the possible explanations for the positive association of body weight with the development of breast cancer. To evaluate the impact of body weight on the risk of breast cancer, we need to consider the role of adipose tissue in the development and differentiation of normal mammary glands. Special attention should be paid to women in their twenties and/or during their lactation periods when the development of normal mammary glands is significant. Further studies are needed to investigate the association between BMI and breast cancer risk, considering the role of body fat in the development of mammary glands.

Cofilin1 controls transcolumnar plasticity in dendritic spines in adult barrel cortex.

During sensory deprivation, the barrel cortex undergoes expansion of a functional column representing spared inputs (spared column), into the neighboring deprived columns (representing deprived inputs) which are in turn shrunk. As a result, the neurons in a deprived column simultaneously increase and decrease their responses to spared and deprived inputs, respectively. Previous studies revealed that dendritic spines are remodeled during this barrel map plasticity. Because cofilin1, a predominant regulator of actin filament turnover, governs both the expansion and shrinkage of the dendritic spine structure in vitro, it hypothetically regulates both responses in barrel map plasticity. However, this hypothesis remains untested. Using lentiviral vectors, we knocked down cofilin1 locally within layer 2/3 neurons in a deprived column. Cofilin1-knocked-down neurons were optogenetically labeled using channelrhodopsin-2, and electrophysiological recordings were targeted to these knocked-down neurons. We showed that cofilin1 knockdown impaired response increases to spared inputs but preserved response decreases to deprived inputs, indicating that cofilin1 dependency is dissociated in these two types of barrel map plasticity. To explore the structural basis of this dissociation, we then analyzed spine densities on deprived column dendritic branches, which were supposed to receive dense horizontal transcolumnar projections from the spared column. We found that spine number increased in a cofilin1-dependent manner selectively in the distal part of the supragranular layer, where most of the transcolumnar projections existed. Our findings suggest that cofilin1-mediated actin dynamics regulate functional map plasticity in an input-specific manner through the dendritic spine remodeling that occurs in the horizontal transcolumnar circuits. These new mechanistic insights into transcolumnar plasticity in adult rats may have a general significance for understanding reorganization of neocortical circuits that have more sophisticated columnar organization than the rodent neocortex, such as the primate neocortex.

Radiation dose rates now and in the future for residents neighboring restricted areas of the Fukushima Daiichi Nuclear Power Plant.

Radiation dose rates were evaluated in three areas neighboring a restricted area within a 20- to 50-km radius of the Fukushima Daiichi Nuclear Power Plant in August-September 2012 and projected to 2022 and 2062. Study participants wore personal dosimeters measuring external dose equivalents, almost entirely from deposited radionuclides (groundshine). External dose rate equivalents owing to the accident averaged 1.03, 2.75, and 1.66 mSv/y in the village of Kawauchi, the Tamano area of Soma, and the Haramachi area of Minamisoma, respectively. Internal dose rates estimated from dietary intake of radiocesium averaged 0.0058, 0.019, and 0.0088 mSv/y in Kawauchi, Tamano, and Haramachi, respectively. Dose rates from inhalation of resuspended radiocesium were lower than 0.001 mSv/y. In 2012, the average annual doses from radiocesium were close to the average background radiation exposure (2 mSv/y) in Japan. Accounting only for the physical decay of radiocesium, mean annual dose rates in 2022 were estimated as 0.31, 0.87, and 0.53 mSv/y in Kawauchi, Tamano, and Haramachi, respectively. The simple and conservative estimates are comparable with variations in the background dose, and unlikely to exceed the ordinary permissible dose rate (1 mSv/y) for the majority of the Fukushima population. Health risk assessment indicates that post-2012 doses will increase lifetime solid cancer, leukemia, and breast cancer incidences by 1.06%, 0.03% and 0.28% respectively, in Tamano. This assessment was derived from short-term observation with uncertainties and did not evaluate the first-year dose and radioiodine exposure. Nevertheless, this estimate provides perspective on the long-term radiation exposure levels in the three regions.

Altered distribution of extracellular matrix proteins in the periodontal ligament of periostin-deficient mice.

Verifying whether periostin affects the distribution of type I collagen, fibronectin and tenascin C in the periodontal ligament (PDL) is important to contribute to a more thorough understanding of that protein's functions. In this study, we have histologically examined incisor PDL of mandibles in 20 week-old male wild-type and periostin-deficient (periostin-/-) mice, by means of type I collagen, fibronectin, tenascin C, proliferating cell nuclear antigen, matrix metallo-proteinase (MMP)-1 and F4/80-positive monocyte/macrophage immunostaining, transmission electron microscopy and quantitative analysis of cell proliferation. Wild-type PDL featured well-arranged layers of collagen bundles intertwined with PDL cells, whose longitudinal axis ran parallel to the collagen fibers. However, cells in the periostin-/- PDL were irregularly distributed among collagen fibrils, which were also haphazardly arranged. Type I collagen and fibronectin reactivity was seen throughout the wild-type PDL, while in the periostin-/- PDL, only focal, uneven staining for these proteins could be seen. Similarly, tenascin C staining was evenly distributed in the wild-type PDL, but hardly seen in the periostin-/- PDL. MMP-1 immunoreactivity was uniformly distributed in the wild-type PDL, but only dotted staining could be discerned in the periostin-/- PDL. F4/80-positive monocyte/macrophages were found midway between tooth- and bone-related regions in the wild-type PDL, a pattern that could not be observed in the periostin-/- PDL. In summary, periostin deficiency may not only cause PDL collagen fibril disorganization, but could also affect the distribution of other major extracellular matrix proteins such as fibronectin and tenascin C.

Fruit and vegetable intake and breast cancer risk defined by estrogen and progesterone receptor status: the Japan Public Health Center-based Prospective Study.

BACKGROUND: Epidemiological evidence for the impact of fruit and vegetable intake on breast cancer risk among the Japanese populations is scarce. OBJECTIVE: The purpose of this study was to evaluate the association between fruit and vegetable intake and breast cancer risk among 47,289 Japanese women. DESIGN: The study was conducted under a population-based prospective cohort design. Dietary assessment was performed using a validated food frequency questionnaire. A Cox proportional hazards regression model was used to calculate relative risks (RRs) and their corresponding 95 % confidence intervals (CIs). RESULTS: During an average of 10.2 years of follow-up, 452 cases of breast cancer were newly diagnosed. No association with breast cancer risk was seen for intake of total fruits and vegetables, cruciferous vegetables, green-leaf vegetables, yellow vegetables, or tomato products in overall or postmenopausal women. Cruciferous vegetable intake was associated with a statistically significant decrease in risk of premenopausal breast cancer [multivariable-RRQ4 vs. Q1 = 0.64 (95 % CI = 0.38-1.10; p trend = .046)] and showed a marginally inverse association with ER+ PR+ tumors [RRper 100 g increment = 0.64 (95 % CI = 0.41-1.00)]. In contrast, positive associations were seen between intake of total fruits and citrus fruits and breast cancer risk in overall and premenopausal women. However, these associations for fruit were all attenuated with additional adjustment for vitamin C intake. CONCLUSIONS: Our results suggest an overall null association between total fruit and vegetable intake and breast cancer risk. Intake of cruciferous vegetable showed a statistically significant association with a decreased risk of breast cancer among premenopausal women.

Impact of body mass index on breast cancer in accordance with the life-stage of women.

A large amount of epidemiological evidence suggests that the impact of body weight on breast cancer risk should be heterogeneous throughout the life-stage of women. At birth, high weight has been positively associated with an increased risk of breast cancer. While, the body mass index (a relative body weight; BMI kg/m(2)) has been inversely associated with breast cancer risk among pre-menopausal women. The inverse trend had been observed in both Western and Asian population, with a relatively lower percentage of obesity and higher percentage of leanness, suggested that the inverse trend could be explained not only by the protective impact due to obesity, but also by the increased risk of breast cancer due to leanness. Among post-menopausal women, however, an elevated BMI has been positively associated with the development of breast cancer, particularly in the cases of estrogen receptor-positive (ER+) and progesterone receptor-positive (PR+) tumors. Currently, the mechanisms underlying the heterogeneous impacts between BMI on breast cancer risk and the life-stage of women remain poorly understood. We reviewed several proposed biological mechanisms that may contribute to the various impacts of relative body weight on breast cancer risk across life-stage. We also discussed the impact of BMI upon the outcome of endocrine therapy, particularly for aromatase inhibitor, in breast cancer patients. To prevent breast cancer incidence and recurrence, the desirable BMI of women may differ throughout their life-stage. To define the underlying mechanism for the various impacts of BMI in the context of breast cancer across various female life stages, further studies will be required.

Immunolocalization of sclerostin synthesized by osteocytes in relation to bone remodeling in the interradicular septa of ovariectomized rats.

This study aimed at elucidating whether estrogen deficiency would affect the synthesis of an osteocyte-derived factor, sclerostin, in the mesial region of alveolar bone. Eight 9-week-old Wistar female rats were ovariectomized (OVX) and eight other rats were Sham-operated (Sham). After 4 weeks, the interradicular septa of mandibular first molar were embedded in paraffin and then histochemically examined. Sclerostin-positive osteocytes were located in the superficial layer of the mesial region of Sham bones, whereas the OVX mesial region showed less sclerostin-reactive osteocytes. There was no significant difference in the distribution of estrogen receptor α and terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling -positive cells in the groups studied. The Sham mesial region featured many osteoclasts, and OVX specimens showed numerous osteoclasts in association with intense immunolabeling of the receptor activator of the nuclear factor kB ligand. Contrary to the observations in Sham specimens, a complex meshwork of cement lines was seen in the OVX mesial region, accompanied by an irregularly distributed osteocytic lacunar-canalicular system. In conclusion, estrogen deficiency appears to inhibit osteocyte-derived sclerostin synthesis in the mesial region of the interradicular septum, in a process that seems to be mediated by accelerated bone remodeling rather than by direct effects on osteocytes.

Neuronal stimulation induces autophagy in hippocampal neurons that is involved in AMPA receptor degradation after chemical long-term depression.

Many studies have reported the roles played by regulated proteolysis in synaptic plasticity and memory, but the role of autophagy in neurons remains unclear. In mammalian cells, autophagy functions in the clearance of long-lived proteins and organelles and in adaptation to starvation. In neurons, although autophagy-related proteins (ATGs) are highly expressed, autophagic activity markers, autophagosome (AP) number, and light chain protein 3-II (LC3-II) are low compared with other cell types. In contrast, conditional knock-out of ATG5 or ATG7 in mouse brain causes neurodegeneration and behavioral deficits. Therefore, this study aimed to test whether autophagy is especially regulated in neurons to adapt to brain functions. In cultured rat hippocampal neurons, we found that KCl depolarization transiently increased LC3-II and AP number, which was partially inhibited with APV, an NMDA receptor (NMDAR) inhibitor. Brief low-dose NMDA, a model of chemical long-term depression (chem-LTD), increased LC3-II with a time course coincident with Akt and mammalian target of rapamycin (mTOR) dephosphorylation and degradation of GluR1, an AMPA receptor (AMPAR) subunit. Downstream of NMDAR, the protein phosphatase 1 inhibitor okadaic acid, PTEN inhibitor bpV(HOpic), autophagy inhibitor wortmannin, and short hairpin RNA-mediated knockdown of ATG7 blocked chem-LTD-induced autophagy and partially recovered GluR1 levels. After chem-LTD, GFP-LC3 puncta increased in spines and in dendrites when AP-lysosome fusion was blocked. These results indicate that neuronal stimulation induces NMDAR-dependent autophagy through PI3K-Akt-mTOR pathway inhibition, which may function in AMPAR degradation, thus suggesting autophagy as a contributor to NMDAR-dependent synaptic plasticity and brain functions.

Leisure-time physical activity and breast cancer risk defined by estrogen and progesterone receptor status--the Japan Public Health Center-based Prospective Study.

OBJECTIVE: The study aims to investigate the association between leisure-time physical activity and breast cancer risk in consideration of tumor estrogen-receptor/progesterone-receptor status. METHODS: We conducted a population-based prospective cohort study among 53,578 women in the Japan Public Health Center-based Prospective Study. Leisure-time physical activity was assessed by self-reported questionnaires. A Cox proportional hazards regression model was used to derive relative risks and 95% confidence intervals. RESULTS: From 1990-1993 to the end of 2007, 652 cases were identified. The breast cancer rates (per 100,000 person-years) in the sedentary groups (≤3 days/month) was 84 in overall, 97 in premenopausal and 75 in postmenopausal women. We observed a statistically significant inverse association between leisure-time physical activity and breast cancer risk (relative risk(≥3 days/week vs. ≤3 days/month)=0.73; 95% confidence interval 0.54-1.00; p(trend) 0.037), particularly in estrogen receptor+progesterone receptor+ (relative risk 0.43; 0.19-1.00; p(trend) 0.022), and this inverse trend was apparent among postmenopausal women (relative risk 0.25; 0.06-1.06; p(trend) 0.041). An inverse trend was also observed between daily total physical activity and postmenopausal estrogen receptor+progesterone receptor+ risk (p=0.046). Among body mass index ≥25 kg/m(2) group, leisure-time physical activity was associated with decreased risk (relative risk(≥1 day/week vs. ≤3 days/month)=0.65; 0.43-0.97; p(trend) 0.033). CONCLUSION: Active participation in leisure-time physical activity may contribute to a decrease in breast cancer risk, particularly for postmenopausal estrogen receptor+progesterone receptor+ tumors.

Body weight at age 20 years, subsequent weight change and breast cancer risk defined by estrogen and progesterone receptor status--the Japan public health center-based prospective study.

Few prospective studies have investigated the association between BMI at age 20 years (BMI20y) and breast cancer risk with consideration to estrogen/progesterone receptor status (ER/PR). We evaluated the association between BMI20y and ER/PR-defined breast cancer risk among 41,594 women in the population-based Japan Public Health Center-based Prospective Study. Anthropometric factors were assessed using self-reported questionnaires. Relative risks (RRs) were estimated by Cox proportional hazards regression models. Through to the end of 2006, 452 breast cancer cases were identified. We observed a statistically significant inverse association between BMI20y and breast cancer incidence [multivariable-adjusted RR for each 5-unit increment 0.75 (95%CI=0.61-0.92)], which was not modified by menopausal or recent BMI status. In contrast, recent BMI and subsequent BMI gain were not associated with increased risk among premenopausal women, but were substantially associated with increased risk among postmenopausal women [corresponding RR(recent BMI)=1.31 (95%CI=1.07-1.59); RR(subsequent BMI gain)=1.32 (95%CI=1.09-1.60)]. In subanalyses by receptor status (∼50% of cases), the observed inverse association of BMI20y with risk was consistent with the result for ER-PR- [0.49 (95%CI=0.27-0.88)], while the observed positive associations of BMI gain with postmenopausal breast cancer risk appeared to be confined to ER+PR+ tumors [corresponding RR(for subsequent BMI gain)=2.24 (95%CI=1.50-3.34)]. Low BMI at age 20 years was substantially associated with an increased risk of breast cancer. In contrast, high recent BMI and subsequent BMI gain from age 20 were associated with increased risk of postmenopausal ER+PR+ tumors.

Immunolocalization of DMP1 and sclerostin in the epiphyseal trabecule and diaphyseal cortical bone of osteoprotegerin deficient mice.

In order to define the osteocytic function in accelerated bone remodeling, we examined the distribution of the osteocytic lacunar-canalicular system (OLCS) and osteocyte-secreting molecules--dentin matrix protein (DMP) 1 and sclerostin--in the epiphyses and cortical bones of osteoprotegerin deficient (OPG(-/-)) mice. Silver impregnation visualized a well-arranged OLCS in the wild-type epiphyses and cortical bone, whereas OPG(-/-) mice had an irregular OLCS in the epiphyses, but well-arranged canaliculi in the cortical bone. DMP1-positive osteocytes were evenly distributed throughout the wild-type epiphyses and cortical bone, as well as the OPG(-/-) cortical bone. However, OPG(-/-) epiphyses revealed weak DMP1-immunoreactivity. Thus, osteocytes appear to synthesize more DMP1 as the OLCS becomes regular. In contrast, sclerostin-immunoreactivity was significantly diminished in the OPG(-/-) epiphyses and cortical bone. In OPG(-/-) epiphyses and cortical bone, triple staining demonstrated few sclerostin-positive osteocytes in the periphery of a thick cell layer of alkaline phosphatase-positive osteoblasts and many tartrate resistant acid phosphatase-positive osteoclasts. Summarizing, the regular distribution of OLCS may affect DMP1 synthesis, while the cellular activities of osteoclasts and osteoblasts rather than the regularity of OLCS may ultimately influence sclerostin synthesis.

Leisure-time physical activity and breast cancer risk by hormone receptor status: effective life periods and exercise intensity.

OBJECTIVE: Physical activity may decrease breast cancer risk. However, it is unclear what intensity of exercise and during which life periods this effect on decreasing risk is efficiently expressed, and whether the associations differ by the estrogen-/progesterone- receptor (ER/PR) status of tumors. We investigated associations between age- and intensity-specific leisure-time physical activity and ER/PR-defined breast cancer risk. METHODS: We conducted a hospital-based case-control study in Nagano, Japan. Subjects were 405 cases newly diagnosed (>99% known ER/PR) from 2001 to 2005, who were age-/area-matched with 405 controls. Activity was assessed with a self-reported questionnaire which considered intensity level (moderate and/or strenuous) at different ages (at 12 and 20 years, and in the previous 5 years). Odds ratios (ORs) and 95% confidence intervals were calculated using logistic regression. RESULTS: Strenuous but not moderate physical activity at age 12 was inversely associated with pre- and postmenopausal breast cancer risk across ER/PR subtypes [overall OR(≥ 5 days/week vs. none) = 0.24 (0.14-0.43)]. Moderate physical activity in the previous 5 years was significantly associated with a decrease in risk for postmenopausal ER + PR + tumors only [OR(≥ 1 day/week vs. none) = 0.35 (0.18-0.67)]. CONCLUSION: Strenuous activity in teens and moderate activity after menopause may contribute to a reduction in breast cancer risk.

Ultrastructural observation on cells meeting the histological criteria for preosteoblasts--a study in the mouse tibial metaphysis.

Preosteoblasts are currently defined as the precursors of mature osteoblasts. These cells are morphologically diverse and may represent a continuum during osteoblast differentiation. We have attempted to categorize the different preosteoblastic phenotypes in vivo by examining bone cells expressing the runt-related transcription factor 2, alkaline phosphatase and BrdU incorporation - histological traits of a preosteoblast - under transmission electron microscopy (TEM). TEM observations demonstrated, at least, in part two preosteoblastic subtypes: (i) a cell rich in cisterns of rough endoplasmic reticulum (rER) with vesicles and vacuoles and (ii) a subtype featuring extended cytoplasmic processes that connect with distant cells, with a small amount of scattered cisterns of rER and with many vesicles and vacuoles. ER-rich cells, whose cellular machinery is similar to that of an osteoblast, were often seen adjacent to mature osteoblasts, and therefore, may be ready for terminal differentiation. In contrast, ER-poor and vesicle-rich cells extended their cytoplasmic processes to mature osteoblasts and, frequently, to bone-resorbing osteoclasts. The abundant vesicles and vacuoles identified in this cell type indicate that this cell is involved in vesicular transport rather than matrix synthesis activity. In summary, our study verified the morphological diversity and the ultrastructural properties of osteoblastic cells in vivo.

FGFR3 down-regulates PTH/PTHrP receptor gene expression by mediating JAK/STAT signaling in chondrocytic cell line.

The signaling axis comprising the parathyroid hormone (PTH)-related peptide (PTHrP), the PTH/PTHrP receptor and the fibroblast growth factor receptor 3 (FGFR3) plays a central role in chondrocyte proliferation. The Indian hedgehog (IHH) gene is normally expressed in early hypertrophic chondrocytes, and its negative feedback loop was shown to regulate PTH/PTHrP receptor signaling. In this study, we examined the regulation of PTH/PTHrP receptor gene expression in a FGFR3-transfected chondrocytic cell line, CFK2. Expression of IHH could not be verified on these cells, with consequent absence of hypertrophic differentiation. Also, expression of the PTH/PTHrP receptor (75% reduction of total mRNA) and the PTHrP (50% reduction) genes was reduced in CFK2 cells transfected with FGFR3 cDNA. Interestingly, we verified significant reduction in cell growth and increased apoptosis in the transfected cells. STAT1 was detected in the nuclei of the CFK2 cells transfected with FGFR3 cDNA, indicating predominance of the JAK/STAT signaling pathway. The reduction in PTH/PTHrP receptor gene in CFK2 cells overexpressing FGFR3 was partially blocked by treatment with an inhibitor of JAK3 (WHI-P131), but not with an inhibitor of MAPK (SB203580) or JAK2 (AG490). Altogether, these findings suggest that FGFR3 down-regulates PTH/PTHrP receptor gene expression via the JAK/STAT signaling in chondrocytic cells.