Is creatine kinase responsible for fatigue? Studies of isolated skeletal muscle deficient in creatine kinase.

Creatine kinase (CK) is a key enzyme for maintaining a constant ATP/ADP ratio during rapid energy turnover. To investigate the role of CK in skeletal muscle fatigue, we used isolated whole muscles and intact single fibers from CK-deficient mice (CK(-/-)). With high-intensity electrical stimulation, single fibers from CK(-/-) mice displayed a transient decrease in both tetanic free myoplasmic [Ca(2+)] ([Ca(2+)](i), measured with the fluorescent dye indo-1) and force that was not observed in wild-type fibers. With less intense, repeated tetanic stimulation single fibers and EDL muscles, both of which are fast-twitch, fatigued more slowly in CK(-/-) than in wild-type mice; on the other hand, the slow-twitch soleus muscle fatigued more rapidly in CK(-/-) mice. In single wild-type fibers, tetanic force decreased and [Ca(2+)](i) increased during the first 10 fatiguing tetani, but this was not observed in CK(-/-) fibers. Fatigue was not accompanied by phosphocreatine breakdown and accumulation of inorganic phosphate in CK(-/-) muscles. In conclusion, CK is important for avoiding fatigue at the onset of high-intensity stimulation. However, during more prolonged stimulation, CK may contribute to the fatigue process by increasing the myoplasmic concentration of inorganic phosphate.     

E-cadherin downregulation in cancer: fuel on the fire?

The development, maintenance and repair of tissue requires an exquisite balance between cell proliferation, cell adhesion and cell motility. Equally, tumour initiation and progression are characterized by not only the abnormal expression of genes involved in cell proliferation and survival but also by genes responsible for the control of cell adhesion and cell motility. Central to the process of cell-cell adhesion in epithelial tissues is E-cadherin. Loss of E-cadherin function in tumours results in the rapid progression of relatively benign adenomas to invasive, metastatic carcinomas. Germline mutation of the E-cadherin gene predisposes to diffuse, poorly differentiated gastric cancer, and its downregulation in sporadic tumours is associated with poor clinical prognosis.     

Does creatine supplementation improve functional capacity in elderly women?

The purpose of this study was to determine the effects of short-term (7 days) oral creatine supplementation (0.3 g.kg(-1)) in elderly women during exercise tests that reflect functional capacity during daily living tasks. We assessed several indices of endurance capacity (1-mile walk test, gross mechanical efficiency, ventilatory threshold, and peak oxygen intake determined during cycle-ergometry) and lower-extremity functional performance (time to complete sit-stand test). Subjects were assigned to a creatine (n = 10; age 67 +/- 6 years) or placebo (n = 6; age 68 +/- 4 years) group. We found a significant improvement only after creatine loading in the sit-stand test (placebo: 9.7 +/- 0.9 seconds for pretest and 9.3 +/- 0.7 seconds for posttest, p > 0.05; creatine: 10.0 +/- 0.7 seconds for pretest and 8.8 +/- 1.1 seconds for posttest). Significance was recorded at p < 0.05 for the interaction effect (group [creatine, placebo] x time [pretest, posttest]). In elderly women, short-term oral creatine supplementation does not improve endurance capacity but increases the ability to perform lower-body functional living tasks involving rapid movements.     

Effects of activin and TGFβ on p21 in colon cancer

Activin and TGFβ share SMAD signaling and colon cancers can inactivate either pathway alone or simultaneously. The differential effects of activin and TGFβ signaling in colon cancer have not been previously dissected. A key downstream target of TGFβ signaling is the cdk2 inhibitor p21 (p21(cip1/waf1)). Here, we evaluate activin-specific effects on p21 regulation and resulting functions. We find that TGFβ is a more potent inducer of growth suppression, while activin is a more potent inducer of apoptosis. Further, growth suppression and apoptosis by both ligands are dependent on SMAD4. However, activin downregulates p21 protein in a SMAD4-independent fashion in conjunction with increased ubiquitination and proteasomal degradation to enhance migration, while TGFβ upregulates p21 in a SMAD4-dependent fashion to affect growth arrest. Activin-induced growth suppression and cell death are dependent on p21, while activin-induced migration is counteracted by p21. Further, primary colon cancers show differential p21 expression consistent with their ACVR2/TGFBR2 receptor status. In summary, we report p21 as a differentially affected activin/TGFβ target and mediator of ligand-specific functions in colon cancer, which may be exploited for future risk stratification and therapeutic intervention.     

Effects of creatine and its analog, β-guanidinopropionic acid, on the differentiation of and nucleoli in myoblasts

The effects of supplementation with creatine (Cr) and its analog, β-guanidinopropionic acid (β-GPA), on the differentiation of myoblasts and the numbers of nucleoli were studied in C2C12 cells. The cells were cultured in differentiation medium for 4 d. Then Cr (1 mM) or β-GPA (1 mM) was added to the cells, and the mixture was cultured for an additional 2 d. Although the number of myotubes was not different among the groups, myotube diameters and nuclear numbers in myotubes were increased by Cr and β-GPA treatment respectively. The expression of differentiation marker proteins, myogenin, and the myosine heavy chain, was increased in the β-GPA group. Supplementation with β-GPA also increased the percentage of p21 (inhibitor for cell cycle progression)-positive myoblasts. Supplementation with Cr inhibited the decrease in nucleoli numbers, whereas β-GPA increased nucleolar sizes in the myotubes. These results suggest that β-GPA supplementation stimulated the differentiation of myoblasts into multi-nucleated myotubes through induction of p21 expression.     

Generation or birth cohort effect on cancer risk in Li–Fraumeni syndrome

Genetic anticipation is the increased incidence, earlier onset, or increased severity of a disease in successive generations. Before the biological basis of anticipation had been demonstrated, the phenomenon was thought to be due to sampling bias, epigenetic effects, gene conversion, or recombinant events. Since then, the biologic basis for anticipation in a number of neurodegenerative disorders has been shown to be attributable to trinucleotide repeat instability, with expansion of repeats clearly correlated with an earlier age of onset. Recently, telomere shortening has been suggested as the mechanism for anticipation in the autosomal dominant form of dyskeratosis congenita, attributable to mutations in the TERC gene, leading to dysfunctional telomeres (Vulliamy et al. 2004). However, the pattern of anticipation has been observed in other disorders, including cancers, for which no genetic defect has been identified. In this study, we assess the apparent generation effect on cancer incidence in ten extended families with P53 germline mutation, identified through probands diagnosed with childhood sarcoma. The probands were from two sets of systematically ascertained sarcoma patients treated at the University of Texas M. D. Anderson Cancer Center between 1944 and 1982. From those overall studies, we have identified ten kindreds having germline P53 mutations in more than one generation. We compared the cancer incidence in members of successive generations of these families with P53 mutations (carriers) and with no P53 mutations (noncarriers). In carriers, cancer incidence increased in succeeding generations; there was no evidence for this effect in noncarriers; however, the noncarrier population was too small to rule it out. The apparent lack of increase in incidence in noncarriers argues against a cohort effect explaining the increase in carriers.     

Barcelona conference on epigenetics and cancer 2016 – beyond cancer genomes

The Barcelona Conference on Epigenetics and Cancer (BCEC) entitled “Beyond Cancer Genomes” took place October 13^th and 14^th 2016 in Barcelona. The 2016 BCEC was the fourth edition of a series of annual conferences coordinated by Marcus Buschbeck and subsequently organized by leading research centers in Barcelona together with B?DEBATE, a joint initiative of BIOCAT and “La Caixa” Foundation. Salvador Aznar-Benitah, Eduard Batlle, and Raúl Méndez from the Institute for Research in Biomedicine in Barcelona selected the 2016 BCEC panel of speakers. As the title indicates, this year's conference expanded the epigenetic focus to include additional cancer-relevant topics, such as tumor heterogeneity and RNA regulation. Methods to develop therapeutic approaches on the basis of novel insights have been discussed in great detail. The conference has attracted 217 participants from 11 countries.     

Effect of thapsigargin on Ca²+ fluxes and viability in human prostate cancer cells

Effect of the carcinogen thapsigargin on human prostate cancer cells is unclear. This study examined if thapsigargin altered basal [Ca2?](i) levels in suspended PC3 human prostate cancer cells by using fura-2 as a Ca2?-sensitive fluorescent probe. Thapsigargin at concentrations between 10?nM and 10 μM increased [Ca2?](i) in a concentration-dependent fashion. The Ca2? signal was reduced partly by removing extracellular Ca2? indicating that Ca2? entry and release both contributed to the [Ca2?](i) rise. This Ca2? influx was inhibited by suppression of phospholipase A2, but not by inhibition of store-operated Ca2? channels or by modulation of protein kinase C activity. In Ca2?-free medium, pretreatment with the endoplasmic reticulum Ca2? pump inhibitor 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ) nearly abolished thapsigargin-induced Ca2? release. Conversely, pretreatment with thapsigargin greatly reduced BHQ-induced [Ca2?](i) rise, suggesting that thapsigargin released Ca2? from the endoplasmic reticulum. Inhibition of phospholipase C did not change thapsigargin-induced [Ca2?](i) rise. At concentrations of 1-10 μM, thapsigargin induced cell death that was partly reversed by chelation of Ca2? with BAPTA/AM. Annexin V/propidium iodide staining data suggest that apoptosis was partly responsible for thapsigargin-induced cell death. Together, in PC3 human prostate cancer cells, thapsigargin induced [Ca2?](i) rises by causing phospholipase C-independent Ca2? release from the endoplasmic reticulum and Ca2? influx via phospholipase A2-sensitive Ca2? channels. Thapsigargin also induced cell death via Ca2?-dependent pathways and Ca2?-independent apoptotic pathways.     

An error catastrophe in cancer?

A comparison between the evolution of cancer cell populations and RNA viruses reveals a number of remarkable similarities. Both display high levels of plasticity and adaptability as a consequence of high degrees of genetic variation. It has been suggested that, as it occurs with RNA viruses, there is a threshold in the levels of genetic instability affordable by cancer cells in order to be able to overcome selection barriers (Trends Genet. 15 (1999) M57). Here we explore this concept by means of a simple mathematical model. It is shown that an error threshold exists in this model, which investigates both competition between cancer cell populations and its impact on overall tumor growth dynamics. Once the threshold is reached, the highly unstable tumor cell populations, which were sustaining malignant growth, become unable to maintain their genetic information, which in turn triggers a slowed down overall tumor growth regime.     

Immunocytochemistry on scrape cytology in breast cancer: will it unearth the weaker positives?

OBJECTIVE: To standardize the technique of immunocytochemical (ICC) assessment of estrogen (ER) and progesterone receptor (PR) status in breast cancer by scrape cytology and to compare the results with immunohistochemistry on paraffin blocks. STUDY DESIGN: ICC assessment for ER and PR was done on scrape smears from tissue samples in 200 cases of primary breast cancer. The results were compared to those obtained from immunohistochemical (IHC) evaluation of formalin-fixed paraffin same tissue samples. RESULTS: ER/PR positivity rates as well as staining scores were compared between the scrape smears and tissue sections. The concordance between cytology and histology was 84% for ER and 90% for PR. Both the positivity rates and the staining intensity scores were higher for cytochemistry than for histochemistry. CONCLUSION: The ICC method on scrape smears is a simple test with rapid turnaround time. The sample required is small, and antigen loss due to fixation and processing is minimal. This new method gives a higher yield of hormone receptor positivity and, when used in conjunction with the IHC method, may improve the pickup rate of ER-positive cases, thereby playing an important role in risk stratification and therapeutic decision making in patients with breast cancer.     

Expressing creatine kinase in transgenic tobacco – a first step towards introducing an energy buffering system in plants

Creatine kinase a key enzyme in cellular energy homeostasis of vertebrates offers the promise of engineering plants with enhanced stress tolerance. In order to provide plants with such an energy buffering system, tobacco was transformed with a cDNA, encoding the cytosolic brain-type isoform of chicken creatine kinase (BB-CK), the expression of which was under the control of the cauliflower mosaic virus 35S (CaMV 35S) promoter. Transgenic tobacco plants were selected and suspension cultures generated. Both transgenic plants and suspension cultures were shown to stably express enzymatically active BB-CK in vitro and in vivo, and in most cases for three successive generations (T₀–T₂). Exogenously supplied creatine was shown to enter the plant cells and resulted in only a slight reduction in root growth at concentrations up to 10mM. Furthermore, the BB-CK expressing tobacco plants and cell suspension cultures were able to convert creatine into phosphocreatine.     

Tumor associated macrophage × cancer cell hybrids may acquire cancer stem cell properties in breast cancer

Breast cancer is one of the most frequently diagnosed cancers among women, and metastasis makes it lethal. Tumor-associated macrophages (TAMs) that acquire an alternatively activated macrophage (M2) phenotype may promote metastasis. However, the underlying mechanisms are still elusive. Here, we examined how TAMs interact with breast cancer cells to promote metastasis. Immunohistochemistry was used to examine the expression of the M2-specific antigen CD163 in paraffin-embedded mammary carcinoma blocks to explore fusion events in breast cancer patients. U937 cells were used as a substitute for human monocytes, and these cells differentiated into M2 macrophages following phorbol 12-myristate 13-acetate (PMA) and M-CSF stimulation. M2 macrophages and the breast cancer cell lines MCF-7 and MDA-MB-231 fused in the presence of 50% polyethylene glycol. Hybrids were isolated by fluorescence-activated cell sorting, and the relevant cell biological properties were compared with their parental counterparts. Breast cancer stem cell (BCSC)-related markers were quantified by immunofluorescence staining, RT-PCR, quantitative RT-PCR and/or western blotting. The tumor-initiating and metastatic capacities of the hybrids and their parental counterparts were assessed in NOD/SCID mice. We found that the CD163 expression rate in breast cancer tissues varied significantly and correlated with estrogen receptor status (p<0.05). The fusion efficiency of either breast cancer cell line with M2 macrophages ranged from 1.81 to 6.47% in the presence of PEG, and no significant difference was observed between the breast cancer cell lines used (p>0.05). Characterization of the fusion hybrids revealed a more aggressive phenotype, including increased migration, invasion and tumorigenicity, but reduced proliferative ability, compared with the parental lines. The hybrids also gained a CD44(+)CD24(-/low) phenotype and over-expressed epithelial-mesenchymal transition-associated genes. These results indicate that TAMs may promote breast cancer metastasis through cell fusion, and the hybrids may gain a BCSC phenotype.     

Unique features of breast cancer in Asian women—Breast cancer in Taiwan as an example

Breast carcinoma is one of the most common cancers in women and is known to arise from a multifactorial process, the effect of reproductive risk factors strongly supporting a hormonal role in its etiology. Breast cancer in Asia is characterized by a lower incidence than in Western populations, but is still the leading type of cancer in Asian women, and a significant increasing tread indicates that it is an issue of particular public health importance. Asian breast cancer is characterized by early tumor onset, showing a relatively younger median age at diagnosis. Recently, scientists began to explore the tumorigenic mechanisms underlying breast cancer formation at the molecular level. Both a candidate-gene approach and genome-wide association studies have yielded crucial insights into breast cancer susceptibility genes initiating breast tumorigenesis. As expected, ethnic/racial variation in the genotypic frequency of these genes results in differences in breast cancer incidence in different populations. Furthermore, the question of how important these genes are in Asian breast cancer remains to be explored.It has been demonstrated that gene expression profiles and gene sets are prognostic and predictive for patients with breast cancer. Originally, due to its early onset, it was speculated that Asian breast cancer would have a higher frequency of the basal-like subtype of breast cancer, a molecular subtype characterized by poor differentiation, resulting in a relatively poor progression; however, recent findings do not support this speculation. The frequency of the luminal-A subtype of breast cancer, characterized by estrogen receptor expression, is similar to that in breast cancer in Caucasian, supporting the usefulness of hormone-based therapy in Asian breast cancer.     

In vivo effects of zoledronic acid on peripheral γδ T lymphocytes in early breast cancer patients

Introduction

Amino-bisphosphonates are potent activators of human γδ T cells. The aim of our study was to evaluate the immunomodulating properties of a single-dose of zoledronic acid (ZA) on γδ T cells in a select group of disease-free breast cancer patients with osteopenia.

Materials and methods

Blood samples were obtained, from 23 patients, before and 7, 28, 56, 90 and 180 days after a single-dose (4 mg) of ZA and analyzed by flow cyometry.

Results

A significant decrease of the different γδ T cell subsets was observed: Naïve (CD3+/Vdelta2+/CD45RA+/CD27+) after 180 days (P < 0.01); Central Memory (CD3+/Vdelta2+/CD45RA-CD27+) after 28 (P < 0.05), 90 (P < 0.01) and 180 days (P < 0.01); and Effector Memory (CD3+/Vdelta2+/CD45RA-/CD27-) after 56 (P < 0.01) and 90 (P < 0.05) days. Based on the observed γδ T cells kinetics patients could be divided in two groups: “responders” that showed a significant decrease in total numbers of γδ T cells and “non-responders” that showed no significant change. However, in vitro phosphoantigen stimulation of patients cells did not show significant differences in terms of IFN-γ response by Vδ2 T cells.

Conclusion

We describe for the first time a long-lasting activation of effector subsets of γδ T cells in disease-free breast cancer patients after a single-dose of ZA. Our results highlight the need to further investigate the clinical significance of the immunomodulating properties of N-BPs.     

Gastric cancer & prospects of cancer in Saudi Arabia peninsula

Gastric cancer is classified to be an aggressive disease with poor treatment outcome, as most cases remain undetected until later stages, wherein surgery and few chemotherapeutics become the only recommended treatment course. The process of cancer development is multistep involving many stages and types of precancerous lesions, and hence, routine monitoring becomes a necessity in those detected with these or exposed to risk factors. Studying the pattern of gastric cancer for any geographical region is also important to control mortality and focus on implementation of efficient management and treatment guidelines. The cause for gastric cancer can be genetic, racial as well as environmental, and hence the pattern of this malignancy differs across geographical regions and between the developing and the developed nations. In case of the Kindgom of Saudi Arabia, very few hospital-based reports have been published highlighting the pattern of gastric cancer, and the associated incidence and mortality rates. However, classified to be one of the most crucial cancer forms in Saudi Arabia, research pertaining to epidemiology, presentation and pathological features are limited. Studying gastric cancer occurrence from public health viewpoint is important also because eradication of causative agents like those that H. pylori has also shown been not reduce the risk of cancer development among individuals with atrophic metaplastic gastritis. In case of Saudi Arabia, many inherent risks for this malignancy exists like waterpipe smoking and shift in diet pattern from the traditional Mediterranean diet. Our review focusses on pattern of gastric cancer on a global scale in comparison to scenario in Saudi Arabia. The aim is to encompass all of the less stressed upon facts about this malignancy in the Kingdom, paving way for future work in this regards.