Cell Journal (Yakhteh)
Volume:13 Issue: 3, Autumn 2011

The neoplastic niche comprises complex interactions between multiple cell types and molecules requiring cell-cell signaling as well as local secretion. These niches are important for both the maintenance of cancer stem cells and the induction of neoplastic cells survival and proliferation. Each niche contains a population of tumor stem cells supported by a closely associated vascular bed comprising mesenchyme-derived cells and extracellular matrix. Targeting cancer stem cells and neoplastic niche may provide new therapies to eradicate tumors. Much progress has been very recently made in the understanding of the cellular and molecular interactions in the microenvironment of neoplastic niches. This review article provides an overview of the neoplastic niches in the bone marrow. In addition to highlighting recent advances in the field, we will also discuss components of the niche and their signaling pathways.

As effectiveness of the autologous graft in the repair of long nerve defects is very limited an effective substitute is needed. This study was conducted to determine the poled polyvinylidene fluoride (PVDF) tube as an alternative to nerve autograft. The left sciatic nerve was transected in 45 male Wistar rats. The animals were then divided randomly into three groups: in an epineural group the nerve was sutured end to end; in an autograft group a 10 mm piece of sciatic nerve was cut, rotated 180° and sutured in the nerve gap; and in a nerve guidance channel group (NGC), PVDF, tube containing nerve growth factor (NGF) and collagen gel was placed in the gap. In a control (n=15) group the sciatic nerve was exposed but not transected. To determine axonal regeneration, retrograde DiI tracer was injected into the gastrocnemius muscle. One week later, retrograde-labeled neurons were counted in the L4-L6 spinal segments and one way ANOVA analysis was performed to compare groups. Neuronal morphology changes were studied by electron microscopy. Significant statistical decreases in the mean number of labeled motoneurons were observed in all surgical groups compared to the control group; and in the autograft and the NGC groups compared to epinural suture group (p<0.01). No significant difference in the mean number of motoneurons was observed between the autograft and NGC groups. Chromatin condensation, dilated endoplasmic reticulum and large vacuoles were observed in the autograft and NGC groups. Regarding the positive effects of PVDF tube containing NGF and Collagen gel on the sciatic nerve regeneration, authors suggest that it may be useful in peripheral nerve repair.

Testicular cell transplantation has been widely used to investigate the restoration of fertility in rodent models. In this research we apply transplantation as a treatment method in the cryptorchid model and compare this method with orchidopexy, which is the routine treatment for this problem. We studied the controversial effects of treatment on the number of germ cells and other morphometrical characteristics of testicular and epididymal parameters in cryptorchid mice. Bilateral cryptorchidism was induced in immature mice by returning two testes to the abdominal cavity via a surgical procedure. Respectively orchidopexy and transplantation of spermatogonial stem cells (were isolated from bilateral cryptorchid testes) with later orchidopexy was performed two and three months after heat exposure in separate cryptorchid mice. The weight of testes, spermatogenic cell numbers, as well as epididymal sperm parameters were measured at two and eight weeks after treatment. The results were analyzed by performing ANOVA and Tukey’s tests. Our results showed that after orchidopexy, the testis remained atrophied and the number of spermatogonia returned to the near normal range, but spermatogenesis was recovered only partially at the stage of differentiated germ cells. After transplantation we observed significant changes in the stage of sperm formation compared to orchidopexy. We demonstrated that the spermatogonia isolated from bilateral cryptorchid mice have the ability to regenerate spermatogenesis. Also, while orchidopexy is a routine treatment for cryptorchidism, transplantation may thus prove to be a promising technique for the preservation of fertility for severely damaged cryptorchid testes that have scarce spermatogonia.

Previous studies, focusing on the effects of abused drugs, have used mice or rats as the main animal models; the present study tries to introduce a simple animal model. For this propose, we investigated the effects of oral morphine consumption by parents on the development of larvae, pupae and imago in Drosophila Melanogaster (D. Melanogaster). In this experimental study, twenty male and 20 female D. Melanogaster pupae were housed in test tubes with banana (5 pupae /tube). Male and female groups each were divided into three experimental group and one control group, which were maintained at 25°C. Morphine (0.2, 0.02, 0.002 mg/ml) was added into the test tubes of the experimental groups. The control group maintained at morphine-free test tube. The male and female groups with the same treatment were coupled and then female fertilization, egg deposit, larval, pupae and imago stages were studied macro and microscopically. The SPSS software (version 9.01) was used for statistical evaluations. In the experimental groups, in the larvae stage, both increase and decrease of length and surface area in the pupae stage were observed. The number of larvae pupae, and imago was reduced in the experimental groups. The study showed that oral morphine consumption by parents may affect the development of larvae, pupation and imago stages in D. Melanogaster. The results also showed that D. Melanogaster may be a reliable animal model to study on the concerns about abused drugs especially those with opioids.

CD44+/CD24-/low breast cancer cells have tumour-initiating properties with stem cell-like features. Breast cancer gene 1 (BRCA1) is a tumour suppressor gene that plays a crucial role in DNA repair and maintenance of chromosome stability. The clinicopathological features of breast cancer in BRCA1 mutation carriers suggest that BRCA1 may function as a stem-cell regulator. In the present experimental study we examined the expression and localization of the BRCA1 protein and investigated the prognostic value as well as its relationship with the putative cancer stem cell (CSC) marker (CD44) in 156 tumour samples from a well-characterized series of unselected breast carcinomas using immunohistochemistry. Statistical analysis of the data was performed using SPSS software version 16 (Chicago, IL, USA). In breast tumours, the loss of nuclear expression was detected in 23 cases (15%), whereas cytoplasmic expression of BRCA1 was observed in 133 breast carcinomas (85%). Altered BRCA1 expression was significantly associated with high grade and poor prognosis breast tumours (p=0.006). We further established an inverse significant correlation between BRCA1 expression levels and CD44+ cancer cell phenotype (p=0.02) Loss of BRCA1 expression is a marker of tumour aggressiveness and correlates with CD44+ tumour cell phenotype. Taken together, the present study supports the idea that the loss of BRCA1 results in persistent errors in DNA replication in breast stem cells and provides targets for additional carcinogenic events.

Resin cements, regardless of their biocompatibility, have been widely used in restorative dentistry during the recent years. These cements contain hydroxy ethyl methacrylate (HEMA) molecules which are claimed to penetrate into dentinal tubules and may affect dental pulp. Since tooth preparation for metal ceramic restorations involves a large surface of the tooth, cytotoxicity of these cements would be more important in fixed prosthodontic treatments. The purpose of this study was to compare the cytotoxicity of two resin cements (Panavia F2 and Rely X Plus) versus zinc phosphate cement (Harvard) using rat L929-fibroblasts in vitro. In this experimental study, ninety hollow glass cylinders (internal diameter 5-mm, height 2-mm) were made and divided into three groups. Each group was filled with one of three experimental cements; Harvard Zinc Phosphate cement, Panavia F2 resin cement and Rely X Plus resin cement. L929- Fibroblast were passaged and subsequently cultured in 6-well plates of 5×105 cells each. The culture medium was RPMI_ 1640. All samples were incubated in CO2. Using enzyme-linked immune-sorbent assay (ELISA) and (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) (MTT) assay, the cytotoxicity of the cements was investigated at 1 hour, 24 hours and one week post exposure. Statistical analyses were performed via two-way ANOVA and honestly significant difference (HSD) Tukey tests. This study revealed significant differences between the three cements at the different time intervals. Harvard cement displayed the greatest cytotoxicity at all three intervals. After 1 hour Panavia F2 showed the next greatest cytotoxicity, but after 24-hours and one-week intervals Rely X Plus showed the next greatest cytotoxicity. The results further showed that cytotoxicity decreased significantly in the Panavia F2 group with time (p<0.005), cytotoxicity increased significantly in the Rely X Plus group with time (p<0.001), and the Harvard cement group failed to showed no noticeable change in cytotoxicity with time. Although this study has limitations, it provides evidence that Harvard zinc phosphate cement is the most cytotoxic product and Panavia F2 appears to be the least cytotoxic cement over time.

Synthetic fluorescent dyes that are conjugated to antibodies are useful tools to probe molecules. Based on dye chemical structures, their photobleaching and photostability indices are quite diverse. It is generally believed that among different fluorescent dyes, Alexa Fluor family has greater photostability than traditional dyes like fluorescein isothiocyanate (FITC) and Cy5. Alexa Fluor 568 is a member of Alexa Fluor family presumed to have superior photostability and photobleahing profiles than FITC. In this experimental study, we conjugated Alexa Fluor 568 and FITC dyes to a mouse anti-human nestin monoclonal antibody (ANM) to acquire their photobleaching profiles and photostability indices. Then, the fluorophore/antibody ratios were calculated using a spectrophotometer. The photobleaching profiles and photostability indices of conjugated antibodies were subsequently studied by immunocytochemistry (ICC). Samples were continuously illuminated and digital images acquired under a fluorescent microscope. Data were processed by ImageJ software. Alexa Fluor 568 has a brighter fluorescence and higher photostability than FITC. Alexa Fluor 568 is a capable dye to use in photostaining techniques and it has a longer photostability when compared to FITC.

Despite of many benefits, umbilical cord blood (UCB) hematopoietic stem cell (HSC) transplantation is associated with low number of stem cells and slow engraftment; in particular of platelets. So, expanded HSCs and co-transfusion of megakaryocyte (MK) progenitor cells can shorten this period. In this study, we evaluated the cytokine conditions for maximum expansion and MK differentiation of CD133+ HSCs. In this experimental study, The CD133+ cells were separated from three cord blood samples by magnetic activated cell sorting (MACS) method, expanded in different cytokine combinations for a week and differentiated in thrombopoietin (TPO) for the second week. Differentiation was followed by the flow cytometry detection of CD41 and CD61 surface markers. Colony forming unit (CFU) assay and DNA analysis were done for colonogenic capacity and ploidy assay. CD133+ cells showed maximum expansion in the stem span medium with stem cell factor (SCF) + FMS-like tyrosine kinase 3-ligand (Flt3-L) + TPO but the maximum differentiation was seen when CD133+ cells were expanded in stem span medium with SCF + Interleukin 3 (IL-3) + TPO for the first and in TPO for the second week. Colony Forming Unit-MK (CFU-MK) was formed in three sizes of colonies in the mega-cult medium. In the DNA analysis; 25.2 ± 6.7% of the cells had more than 2n DNA mass. Distinct differences in the MK progenitor cell count were observed when the cells were cultured in stem span medium with TPO, SCF, IL-3 and then the TPO in the second week. Such strategy could be applied for optimization of CD133+ cells expansion followed by MK differentiation.

The clavulanic acid regulatory gene (claR) is in the clavulanic acid biosynthetic gene cluster that encodes ClaR. This protein is a putative regulator of the late steps of clavulanic acid biosynthesis. The aim of this research is the molecular cloning of claR, isolated from the Iranian strain of Streptomyces clavuligerus (S. clavuligerus). In this experimental study, two different strains of S. clavuligerus were used (PTCC 1705 and DSM 738), of which there is no claR sequence record for strain PTCC 1705 in all three main gene banks. The specific designed primers were subjected to a few base modifications for introduction of the recognition sites of BamHI and ClaI. The claR gene was amplified by polymerase chain reaction (PCR) using DNA isolated from S. clavuligerus PTCC 1705. Nested-PCR, restriction fragment length polymorphism (PCR-RFLP), and sequencing were used for molecular analysis of the claR gene. The confirmed claR was subjected to double digestion with BamHI and ClaI. The cut claR was ligated into a pBluescript (pBs) vector and transformed into E. coli. The entire sequence of the isolated claR (Iranian strain) was identified. The presence of the recombinant vector in the transformed colonies was confirmed by the colony-PCR procedure. The correct structure of the recombinant vector, isolated from the transformed E. coli, was confirmed using gel electrophoresis, PCR, and double digestion with restriction enzymes. The constructed recombinant cassette, named pZSclaR, can be regarded as an appropriate tool for site directed mutagenesis and sub-cloning. At this time, claR has been cloned accompanied with its precisely selected promoter so it could be used in expression vectors. Hence the ClaR is known as a putative regulatory protein. The overproduced protein could also be used for other related investigations, such as a mobility shift assay.

The role of staphylococcal enterotoxin B (SEB) in food poisoning is well known, however its role in other diseases remains to be explored. The aim of this study is the molecular screening and characterization of the SEB gene in clinically isolated strains. In this experimentally study, 300 Staphylococcus aureus (S. aureus) strains isolated from clinical samples were assayed. The isolated strains were confirmed by conventional bacteriological methods. Polymerase chain reaction (PCR) was used to determine the enterotoxin B (ent B) gene. Assessment of toxin production in all strains that contained the ent B gene was then performed. Finally, using specific antibody against SEB, a Western-blot was applied to confirm detection of enterotoxin B production. Results indicated that only 5% of the 300 clinically isolated S. aureus contained the ent B gene. All strains which contained the ent B gene produced a proteinous enterotoxin B. The results of sequence determination of the PCR product were compared with the gene bank database and 98% similarity was achieved. The results of the Western-blot confirmed that enterotoxin B was produced in strains that contained the ent B gene. The results of this study indicate that 5% of clinically isolated S. aureus strains produce enterotoxin B. Considering that the enterotoxin B is an important superantigen, it is possible that a delay in diagnosis and lack of early proper treatment can cause an incidence of late complications, particularly in staphylococcal chronic infections. For this reason, it is suggested that in addition to detecting bacteria, an enterotoxin B detection test should be performed to control its toxigenicity.