New cell or new cycle?

In mammals, trophoblast giant (TG) cell differentiation is characterized by a physiological endoreduplication, resulting in genome size augmentation. A recent study by Ullah and colleagues (pp. 3024-3036), published in this issue of Genes & Development, now elucidates the role of the cyclin-dependent kinase inhibitors (CKIs), p21 and p57, in mammalian endocycle regulation.     

δ-Cadinene inhibits the growth of ovarian cancer cells via caspase-dependent apoptosis and cell cycle arrest

Ovarian cancer is one of the most common causes of mortality among all cancers in females and is the primary cause of mortality from gynecological malignancies. The objective of the current research work was to evaluate a naturally occurring sesquiterpene-δ-Cadinene for its antiproliferative and apoptotic effects on human ovary cancer (OVCAR-3) cells. We also demonstrated the effect of δ-Cadinene on cell cycle phase distribution, intracellular damage and caspase activation. Sulforhodamine B (SRB) assay was used to evaluate the antiproliferative effect of δ-cadinene on OVCAR-3 cells. Cellular morphology after δ-cadinene treatment was demonstrated by inverted phase contrast microscopy, fluorescence microscopy and transmission electron microscopy. Flow cytometry was used to analyze the effect of δ-cadinene on cell cycle phase distribution and apoptosis using propidium iodide and Annexin V-fluorescein isothiocyanate (FITC)/PI kit. The results revealed that δ-cadinene induced dose-dependent as well as time-dependent growth inhibitory effects on OVACR-3 cell line. δ-cadinene also induced cell shrinkage, chromatin condensation and nuclear membrane rupture which are characteristic of apoptosis. Treatment with different doses of δ-cadinene also led to cell cycle arrest in sub-G1 phase which showed dose-dependence. Western blotting assay revealed that δ-cadinene led to activation of caspases in OVCAR-3 cancer cells. PARP cleavage was noticed at 50 µM dose of δ-cadinene with the advent of the cleaved 85-kDa fragment after exposure to δ-cadinene. At 100 µM, only the cleaved form of PARP was detectable. Pro-caspase-8 expression remained unaltered until 10 µM dose of δ-cadinene. However, at 50 and 100 µM dose, pro-caspase-8 expression was no longer detectable. There was a significant increase in the caspase-9 expression levels after 50 and 100 µM δ-cadinene treatments.     

Power output during women’s World Cup road cycle racing

Little information exists on the power output demands of competitive women’s road cycle racing. The purpose of our investigation was to document the power output generated by elite female road cyclists who achieved success in FLAT and HILLY World Cup races. Power output data were collected from 27 top-20 World Cup finishes (19 FLAT and 8 HILLY) achieved by 15 nationally ranked cyclists (mean ± SD; age: 24.1±4.0 years; body mass: 57.9±3.6 kg; height: 168.7±5.6 cm; 63.6±2.4 mL kg⁻¹ min⁻¹; peak power during graded exercise test (GXT_{peak power}): 310±25 W). The GXT determined GXT_{peak power}, lactate threshold (LT) and anaerobic threshold (AT). Bicycles were fitted with SRM powermeters, which recorded power (W), cadence (rpm), distance (km) and speed (km h⁻¹). Racing data were analysed to establish time in power output and metabolic threshold bands and maximal mean power (MMP) over different durations. When compared to HILLY, FLAT were raced at a similar cadence (75±8 vs. 75±4 rpm, P=0.93) but higher speed (37.6±2.6 vs. 33.9±2.7 km h⁻¹, P=0.008) and power output (192±21 vs. 169±17 W, P=0.04; 3.3±0.3 vs. 3.0±0.4 W kg⁻¹, P=0.04). During FLAT races, riders spent significantly more time above 500 W, while greater race time was spent between 100 and 300 W (LT-AT) for HILLY races, with higher MMPs for 180–300 s. Racing terrain influenced the power output profiles of our internationally competitive female road cyclists. These data are the first to define the unique power output requirements associated with placing well in both flat and hilly women’s World Cup cycling events.     

Excitability of the soleus reflex arc during intensive stretch–shortening cycle exercise in two power-trained athlete groups

In several explosive types of sport events the leg extensor muscles are subjected to very high impact loads. Thus, extreme requirements exist for the neuromuscular system to develop sufficient muscle stiffness in the lower extremities in order to tolerate these high impact loads. Therefore, it would be challenging to measure reflex modulation during high impact activities, and with different athlete populations. In the present experiment, H-reflex and short latency reflex (M1) sensitivity was measured during drop jump exercises among high jumpers and sprinters. The changes in both reflex peak-to-peak amplitudes showed a significant (P < 0.05) reduction towards the end of the exercise for the sprinters. In addition, the same subject group showed a remarkable increase in serum creatine kinase (CK) activity 2 h after the jumps. Similar changes could not be observed for the high jumpers. These results clearly indicate different neural adaptation strategies for the two athlete groups. Reduction in H-reflex sensitivity and an increase in CK-activity in sprinters were taken as evidence for presynaptic inhibition, probably induced by substances related to muscle damage. Since high jump training includes more high impact loading, it was assumed that it could lead to some structural adaptation and, thus, prevents exercise induced reflex modification to a certain extent.     

Transforming growth factor-β1 stimulates mesenchymal stem cell proliferation by altering cell cycle through FAK-Akt-mTOR pathway

Background: Mesenchymal stem cells (MSCs) are promising for cell therapy and regenerative medicine. An increased need for expanding of MSCs under serum-free condition to achieve a sufficient quantity for therapeutic applications is inevitable. Transforming growth factor-β1 (TGF-β1) is widely used for expanding clinical-grade MSCs in vitro. This work focuses on the influence of TGF-β1 on proliferation in rat bone marrow-derived MSCs (BMSCs) and the underlying mechanism.

Materials and Methods: BMSCs were isolated and cultured with or without TGF-β1 in a serum-free medium and Cell Counting Kit-8 assay was used to detect BMSCs proliferation. Cell cycle transition was also analyzed. Further, the expression levels of cyclin D1, phosphorylated focal adhesion kinase, and downstream effectors in Akt-mTOR-S6K1 signaling pathway were examined by western blotting.

Results and Conclusion: TGF-β1 triggered proliferation via accelerating G1/S cell cycle transition in BMSCs. The addition of TGF-β1 can activate Akt-mTOR-S6K1 pathway. Additionally, FAK was found to be involved in the process. Upon adding the FAK inhibitor, both the activation of Akt-mTOR-S6K1 and TGF-β1-induced cell proliferation were abrogated. Together, an insight understanding of how TGF-β1 influences BMSCs proliferation is achieved. This study provides a possible strategy of supplementing TGF-β1 in serum-free medium for in vitro expansion, which eventually would advance the production of clinical-grade MSCs for regenerative medicine.     

Dendritic cell–epithelial cell crosstalk in the gut

Intestinal epithelial cells are fundamental to maintain barrier integrity and to participate in food degradation and absorption, but they can also decipher signals coming from the outside world and ‘educate’ the immune system accordingly. In particular, they interact with dendritic cells (DCs) and other intraepithelial immune cells to drive tolerogenic responses under steady state, but they can also release immune mediators to recruit inflammatory cells and to elicit immunity to infectious agents. When these interactions are deregulated, immune disorders can develop. In this review, we discuss some important features of epithelial cells and DCs and their fruitful interactions.     

Shortening of complementarity determining region 3 of the T cell receptor α chain during thymocyte development

TCR diversity depends mainly on the hypervariable complementarity determining region 3 (CDR3) α and β loops generated by non-germline-encoded nucleotide insertions and/or deletions. It is known that the length of the CDR3β sequence shortens during the process of thymocyte development and that the extent of this shortening is strongly affected by germline-encoded Vβ segments or MHC haplotypes. To examine whether CDR3α shortens to the same extent as CDR3β and how it is affected by Vα segments or MHC haplotypes, we analyzed CDR3α length distributions in thymic CD4+CD8+ (DP), CD4+CD8− (CD4SP) and CD4−CD8+ (CD8SP) T cells of different strains of mice (C57BL/6, C.B10 and Balb/c). As expected, CDR3α shortening occurred in both the CD4SP and CD8SP cells of all strains tested and the extent of shortening varied considerably depending on Vα segment use. However, there was no correlation in the extent of CDR3α shortening in individual Vα segments between CD4SP and CD8SP cells. Interestingly, there was a significant correlation in the extent of CDR3α shortening among different strains of mice only in CD4SP but not CD8SP cells, independent of MHC haplotype. These results suggest that the extent of CDR3α shortening is primarily determined by germline-encoded Vα segments and affected by allelic variation of MHC class I but not of MHC class II. The present study showed that T cells with shorter CDR3α sequences are preferably selected for the functional TCR repertoire during thymocyte development, and this provides an intriguing insight into the interactions of the TCR and p-MHC ligand.     

Role of tumor necrosis factor-α in the regulation of keratinocyte cell cycle and DNA repair after ultraviolet-B radiation

Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine produced in the skin in response to ultraviolet B radiation (UVB). TNF-α facilitates UVB-induced apoptosis and probably contributes to removal of damaged cells. Surprisingly, murine TNF-α-knockout models have demonstrated that TNF-α is necessary for the early stages of skin carcinogenesis and development of squamous cell carcinoma. In the present PhD thesis, we examined the effects of TNF-α on DNA repair and cell cycle regulation in UVB-irradiated keratinocytes. In the model of premalignant keratinocytes (HaCaT), TNF-α abolished the UVB-induced G2/M checkpoint and diminished the DNA repair despite induction of apoptosis. TNF-α activated the protein kinase B/Akt and regulation of its downstream targets, mTOR, Bad and FoxO3a. This effect was dependent on atypical protein kinase C species (aPKC) since a specific peptide blocking the activity of the PKCξ and ι/λ abrogated the activation of Akt by TNF-α. The aPKC-Akt axis was likely to be responsible for the TNF-α-induced decrease in DNA repair since blocking of Akt activity restored DNA repair. Since anti-TNF-α approaches are increasingly used in the therapy of autoimmune diseases and one of the safety concerns is the potential enhancement of skin carcinogenesis, we investigated the effect of the chimeric monoclonal anti-TNF-α antibody infliximab on UVB-irradiated HaCaT cells. Cells treated with infliximab had significantly increased levels of DNA damage despite enhanced G2/M checkpoint arrest, increased apoptosis and inhibition of Akt. In conclusion, we identified a possible novel mechanism by which TNF-α promotes UVB-induced skin carcinogenesis. This depends on aPKC-Akt activation and inhibition of DNA repair. TNF-α-treated cells are prone to escape checkpoint control and are possibly more likely to accumulate mutations, which may constitute a relevant mechanism enhancing tumor development. The effect of anti-TNF-α therapy on skin carcinogenesis warrants further investigation as our study indicates that, in contrast to what had been expected, infliximab may impair DNA repair.     

Stable serum levels of anti-Müllerian hormone during the menstrual cycle: a prospective study in normo-ovulatory women

BACKGROUND: Anti-Müllerian hormone (AMH), secreted by the granulosa cells of preantral and small antral follicles, has been described as a potential marker of the ovarian reserve. The aim of this prospective study is to investigate the variations of AMH during the menstrual cycle in a young selected population of normo-ovulatory women and to analyse the correlation with other cyclic hormones. METHODS: Twenty healthy volunteers from 19 to 35 years old, with regular menstrual cycles (26-31 days), normal ovulation (day 10-16), normal hormonal profile and normal body mass index (18-26 kg/m2) were recruited. AMH, inhibin B, LH, FSH, estradiol and progesterone were measured on days 3, 7, 10, 11, 12, 13, 14, 15, 16, 18, 21 and 25 of a spontaneous cycle. RESULTS: AMH serum levels, either expressed by cycle day or aligned according to the ovulation day, did not show any significant variations during the menstrual cycle. CONCLUSIONS: No significant fluctuation of the AMH level during the menstrual cycle was observed. Therefore, this hormone is particularly interesting for clinical evaluation of the ovarian reserve as it may be used at any time during the cycle.     

Is the menstrual cycle affecting the skin prick test reactivity?

Allergen skin prick tests (SPT) are very sensitive and specific tests to detect allergic sensitization in atopic patients. Certain factors like antihistamines, antidepressant therapies or circadian rhythms can alter the results of SPT. In women, the changes in endogenous hormone levels throughout the menstrual cycle may affect the allergic responses and natural course of allergic diseases. The aim of this study was to investigate the probable influence of the phases of the menstrual cycle on SPT reactivity to allergen extracts and histamine. Forty-two female patients with seasonal allergic rhinoconjunctivitis were enrolled in the study. Skin prick test reactivities to allergens and histamine were measured at the beginning of the menstrual cycle (3rd or 4th day), mid-cycle (14th or 15th day) and end-cycle (27th or 28th day) consecutively. Serum estradiol, progesterone, luteinizing hormone (LH), and follicle stimulating hormone (FSH) levels were determined simultaneously. We observed the most significant reactions to allergens when SPT is performed at mid-cycle. However, SPT reactivity to histamine did not vary throughout the menstrual cycle. Serum estradiol and LH levels showed positive correlation with SPT reactivity to allergens at mid-cycle. Our results suggest that SPT give the best results when they are performed at mid-cycle. Additionally, allergens seem to cause mast cell degranulation to a greater extent in subjects in which endogenous hormones like estradiol and LH are elevated.     

Expression level of the mitotic checkpoint protein and G2–M cell cycle regulators and prognosis in gastrointestinal stromal tumors in the stomach

The biological behavior of gastrointestinal stromal tumors (GISTs) ranges from benign to malignant, and the risk of an adverse outcome is correlated with the location of the primary tumor, tumor size, and mitotic counts. Cell cycle regulators are potentially associated with the tumorigenesis and progression of GISTs. Checkpoint with forkhead and ring finger (CHFR) functions as an important checkpoint protein in the early to mid-prophase to regulate mitosis. In this study, we evaluated the expression of CHFR and several cell cycle regulators, including cyclin A, cyclin B1, cdc2, and cdk2, by immunohistochemical staining in 53 cases of primary gastric GISTs, and compared the immunohistochemical results with the clinicopathological factors or the GIST risk grades as modified by Miettinen et al. Of the 53 cases, 18 (34%) showed decreased nuclear CHFR expression. Decreased CHFR expression was correlated with higher mitotic counts [>5/50 high-power fields (HPFs)] (p = 0.039) and a high-risk grade (p = 0.0475), but not with expression of other cell cycle regulators. Higher cyclin A labeling index (LI, >1.5%), cyclin B1 LI (>0.25%), cdc2 LI (>1.16%), Ki-67 LI (>4.9%), mitotic counts (>5/50 HPF) and high-risk grade were each associated with shorter disease-free survival (p = 0.0017, p = 0.003, p = 0.0471, p = 0.002, p < 0.001, and p = 0.0017, respectively). Our results suggest that modified risk grade and increased expression of G2–M regulators such as cyclin A, cyclin B1, and cdc2 are useful for predicting the biological behavior of gastric GISTs. In addition, decreased CHFR expression may play a role in increased proliferative activity of higher grade GISTs.     

Differentiation of endometrial stromal cells in vitro: down-regulation of suppression of the cell cycle inhibitor p57 by HOXA10?

Decidualization is a critical step during embryo implantation that is characterized by the differentiation of endometrial stromal cells (ESC) into decidua cells. However, the mechanism of differentiation remains largely unknown. Previously, it has been shown that the null function of homeo box A10 (HOXA10) causes defects in both implantation and decidualization, suggesting that the HOXA10 signalling pathway is likely to be involved in uterine decidualization. In the present study, we determined the expression and subcellular distribution of HOXA10 and its downstream molecule, p57, in ESC during in vitro decidualization induced by a combination of 8-bromo-cAMP and medroxyprogesterone acetate. We demonstrated that the HOXA10 was down-regulated while in contrast, p57 was up-regulated in the process of decidualization. Immunocytochemistry and transient expression of the HOXA10 tagged with green fluorescence protein revealed that there were no differences in the HOXA10 subcellular localization between the induced and non-induced ESC. Our results suggest that the down-regulation of HOXA10 may contribute to increased p57 and that up-regulation of p57 likely plays an important role in ESC differentiation in the process of decidualization. The progesterone receptor pathway may participate in promoting ESC to exit the cell cycle and enter differentiation.