Adenoviral Vector-mediated Gene Transfer: Timing of Wild-type P53 Gene Expression in vivo and Effect of Tumor Transduction on Survival in a Rat Glioma Brachytherapy Model

Objective. This study sought to investigate modification of the radiation response in a rat 9L brain tumor model in vivo by the wild-type p53 gene (wtp53). Determination of the timing and dose of radiation therapy required the assessment of the duration of the effect of wtp53 expression on 9L tumors after in vivo transfection. Methods. Anesthetized male F-344 rats each were stereotactically inoculated with 4 × 10⁴ 9L gliosarcoma cells through a skull screw into the cerebrum in the right frontal region. Twelve-day-old tumors were inoculated through the screw with recombinant adenoviral vectors under isoflurane anaesthesia: control rats with Ad5/RSV/GL2 (carrying the luciferase gene), and study rats with Ad5CMV-p53 (carrying the wtp53 gene). Brain tumors removed at specific times after transfection were measured, homogenized, and lysed and wtp53 expression determined by Western blot analysis. Four groups of nine rats were, subsequently, implanted with iodine-125 seeds 15 days post-tumor inoculation to give a minimum tumor dose of 40 or 60Gy. Results. We demonstrated transfer of wtp53 into rat 9L tumors in vivo using the Ad5CMV-p53 vector. The expression of wtp53 was demonstrated to be maximum between days 1 and 3 post-vector inoculation. Tumors expressing wtp53 were smaller than controls transfected with Ad5/RSV/GL2 but this difference was not statistically significant. Radiation made a significant difference to the survival of tumor-bearing rats. Moreover, wtp53 expression conferred a significant additional survival advantage. Conclusion. The expression of wtp53 significantly improves the survival of irradiated tumor-bearing rats in our model.     

Current concepts in the molecular genetics of pediatric brain tumors: implications for emerging therapies

Background The revolution in molecular biology that has taken place over the past 2 decades has provided researchers with new and powerful tools for detailed study of the molecular mechanisms giving rise to the spectrum of pediatric brain tumors. Application of these tools has greatly advanced our understanding of the molecular pathogenesis of these lesions.Review After familiarizing readers with some promising new techniques in the field of oncogenomics, this review will present the current state of knowledge as it pertains to the molecular biology of pediatric brain neoplasms. Along the way, we hope to highlight specific instances where the detailed mechanistic knowledge acquired thus far may be exploited for therapeutic advantage.     

Inhibition of angiogenesis by blocking activation of the vascular endothelial growth factor receptor 2 leads to decreased growth of neurogenic sarcomas.

Neurogenic sarcomas are incurable, common malignant human peripheral nerve tumors subject to local recurrence and systemic metastasis. In this study, the vascularity, vascular endothelial growth factor (VEGF) expression, and effects of inhibiting VEGF receptor on growth of neurogenic sarcomas were examined. Vascularization and VEGF expression were 6.4- and 15-fold higher in tumors than in normal nerves. The small molecule inhibitor (SU5416) of VEGF receptor 2 had no effect on neurogenic sarcoma cell lines in vitro, but the growth of a human tumor explant xenograft model was reduced by 54.8% compared to vehicle. Reduction in tumor growth was due to decreased tumor angiogenesis, leading to reduction of tumor cell proliferation and increased apoptosis. Inhibiting VEGF function may therefore be a useful adjuvant therapy for neurogenic sarcomas.     

Molecular pathways triggering glioma cell invasion

The efficacy of treating malignant gliomas with adjuvant therapies remains largely unsuccessful due to the inability to effectively target invading cells. Although our understanding of glioma oncogenesis has steadily improved, the molecular mechanisms that mediate glioma invasion are still poorly understood. It is clear that genetic alterations in malignant gliomas affect cell proliferation and cell cycle control, which are the targets of most chemotherapeutic agents. However, effective therapy against cell invasion has been less successful. Future treatment protocols must incorporate pharmacotherapeutic strategies that target resistant infiltrative glioma cells as well as proliferating ones. Thus, delineating the point of convergence of signaling pathways, which mediate glioma invasion, proliferation and apoptosis, may identify novel targets that can serve as possible points of therapeutic intervention. The optimization of novel strategies will require reliable preclinical testing using an in vivo animal model of brain invasion. Current applications of existing animal models are not currently optimized or characterized for use in glioma invasion research. As such, the development of a bona fide brain invasion model in vivo must be established. Progress in understanding molecular mechanisms driving glioma invasion will be critical to the success of managing and improving the outcome of patients with this grave disease.     

Analysis of mammalian septin expression in human malignant brain tumors

Septins are a highly conserved subfamily of GTPases that play an important role in the process of cytokinesis. To increase our understanding of the expression and localization of the different mammalian septins in human brain tumors, we used antibodies against septins 2, 3, 4, 5, 6, 7, 9, and 11 in immunofluorescence and Western blot analyses of astrocytomas and medulloblastomas. We then characterized the expression and subcellular distribution of the SEPT2 protein in aphidicolin-synchronized U373 MG astrocytoma cells by immunofluorescence and fluorescence-activated cell sorter analysis. To determine the role of SEPT2 in astrocytoma cytokinesis, we inducibly expressed a dominant-negative (DN) SEPT2 mutant in U373 MG astrocytoma cells. We show variable levels and expression patterns of the different septins in brain tissue, brain tumor specimens, and human brain tumor cell lines. SEPT2 was abundantly expressed in all brain tumor samples and cell lines studied. SEPT3 was expressed in medulloblastoma specimens and cell lines, but not in astrocytoma specimens or cell lines. SEPT2 expression was cell cycle-related, with maximal levels in G2-M. Immunocytochemical analysis showed endogenous levels of the different septins within the perinuclear and peripheral cytoplasmic regions. In mitosis, SEPT2 was concentrated at the cleavage furrow. By immunocytochemistry and flow cytometry, we show that a DN SEPT2 mutant inhibits the completion of cell division and results in the accumulation of multinucleated cells. These results suggest that septins are variably expressed in human brain tumors. Stable expression of the DN SEPT2 mutant leads to a G2-M cell cycle block in astrocytoma cells.     

Inhibition of Rho-kinase affects astrocytoma morphology, motility, and invasion through activation of Rac1

Malignant astrocytomas are highly invasive neoplasms infiltrating diffusely into regions of normal brain. Whereas the molecular and cellular mechanisms governing astrocytoma invasion remain poorly understood, evidence in other cell systems has implicated a role for the Rho-GTPases in cell motility and invasion. Here, we examine how the inhibition or activation of Rho-kinase (ROCK) affects astrocytoma morphology, motility, and invasion. ROCK was inhibited in astrocytoma cells by using 5 to 100 mumol/L of Y27632 or by expressing the dominant-negative ROCK mutant, RB/PH TT. ROCK activation was achieved by expressing a constitutively active mutant, CAT. ROCK inhibition led to morphologic and cytoskeletal alterations characterized by an increase in the number and length of cell processes, increased membrane ruffling, and collapse of actin stress fibers. Using two-dimensional radial migration and Boyden chamber assays, we show that astrocytoma migration and invasion were increased at least 2-fold by ROCK inhibition. On the contrary, ROCK activation significantly inhibited migration and invasion of astrocytoma cells. Furthermore, using a Rac-GTP pull-down assay, we show that Rac1 is activated as a consequence of ROCK inhibition. Finally, we show that treatment of astrocytoma cells with small interfering RNA duplexes specific for Rac1-reversed stellation, prevented membrane ruffling formation and abrogated the increased motility observed following treatment with Y27632. Our data show that Rac1 plays a major role in astrocytoma morphology, motility, and invasion. These findings warrant further investigation to determine precisely how the modulation of Rac1 and ROCK can be exploited to inhibit glioma invasion.     

Evaluation of proliferative index and cell cycle protein expression in choroid plexus tumors in children

Choroid plexus tumors are papillary neoplasms originating from the epithelium of the choroid plexus within the cerebral ventricles. They may be highly proliferative tumors, but detailed studies confirming their proliferative potential are lacking. Accordingly, we performed a clinicopathological correlation study of neoplasms arising from the choroid plexus in children using immunohistochemistry to characterize both their proliferative potential and their degree of cell cycle dysregulation when compared to non-neoplastic choroid epithelium. Twelve children with choroid plexus papillomas (CPPs) and 11 with choroid plexus carcinomas (CPCs) were identified from the time period 1982-1997. The outcome and survival of these children following treatment was determined from the medical record. Immunohistochemical studies were performed on CPPs and CPCs in this patient population and on non-neoplastic choroid epithelium using antibodies to MIB-1, p53, cyclin E, retinoblastoma protein (pRB), p107, and E2F-1. In 5 children with CPCs, tumor tissue was available for immunohistochemistry at a second surgery after cycles of chemotherapy had been given. The mean survival for patients with CPPs was 8.5 years, and with CPCs 5.2 years with a minimum follow-up of 4 years for the group. The expression of cell cycle markers and MIB-1 was greater in CPCs than in CPPs or normal choroid plexus. The expression of MIB-1, p53, pRB, and E2F-1 was significantly lower in patients with CPCs after chemotherapy than before. The MIB-1 labeling index for CPC patients who are alive and well after treatments was 15.19+/-3.2 compared to 22.63+/-3.04 for patients who have died from their disease (P<0.05). We conclude that CPCs in children are characterized by a higher MIB-1 labeling index and greater cell cycle dysregulation than are CPPs. Chemotherapy may work in part on CPCs to decrease their proliferative potential and expression of cell cycle regulatory proteins.     

Biodegradation during contaminant transport in porous media: 7. Impact of multiple-degrader community dynamics

The biodegradation and transport of phenanthrene in porous media containing multiple populations of phenanthrene degraders is examined with a series of miscible-displacement experiments. A long-term experiment was conducted with a soil containing an indigenous microbial community comprised of 25 identified phenanthrene-degrading isolates. The rate and magnitude of phenanthrene biodegradation oscillated throughout the six-month experiment. This behavior, at least in part, is attributed to multiple-population dynamics associated with the indigenous community of phenanthrene degraders, the composition of which changed during the experiment. This hypothesis is supported by the results of experiments conducted using sterilized porous media that were inoculated with selected isolates obtained from the indigenous soil community. The results of experiments conducted with sterilized soil inoculated with isolate A exhibited an initial extended period of steady phenanthrene effluent concentrations, followed by a uniform decline. The results of experiments conducted using sterilized sand for single-isolate systems with one of three selected isolates and for systems of two-isolate combinations, indicate the existence of apparent synergistic and antagonistic interactions among the isolates. For example, phenanthrene biodegradation was relatively extensive and occurred without a lag phase for isolate A alone. However, biodegradation was constrained when isolate A and B were combined, indicating an antagonistic interaction. Conversely, whereas extensive lag phases were exhibited by both isolates B and C for the single-isolate experiments, there was minimal lag when isolates B and C were combined, indicating a synergistic interaction.     

Systemic review of the epidemiology of autism in Arab Gulf countries

Objective:

To assess the current state of knowledge on the epidemiology of autism in Arab Gulf countries, and identify gaps for future research.

Methods:

PubMed and ScienceDirect databases were used to identify relevant articles published until the 3rd of April 2013 (date of search). The search was conducted using the electronic library of King Saud Bin Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia. Studies were eligible for inclusion if they concerned the epidemiology of autism, conducted in any Arab Gulf country, and published in English.

Results:

Twelve articles met the inclusion criteria. Studies showed a prevalence ranging from 1.4 to 29 per 10,000 persons. Identified risk factors were metabolic, autoimmune, and environmental in nature. The following determinants were found as possible contributing factors for autism: suboptimal breast-feeding, advanced maternal and paternal age, cesarean section, and prenatal complications.

Conclusion:

Only a few studies explored the epidemiology of autism in Arab Gulf countries and none have investigated the burden of the disease on the child, family, or society. More research is needed to better identify the burden and risk factors of autism in Gulf countries.Autism or autistic disorder is a neurodevelopmental disorder that impairs a person’s ability to communicate, interact socially with others, and respond to certain stimuli in their surroundings. The condition is usually diagnosed by the age of 3 and is more prevalent in males than females.1 Other closely related terminologies are autism spectrum disorders (ASD), and pervasive developmental disorders (PDD). Autism spectrum disorder is characterized by delayed language development, repetitive and stereotyped patterns of behavior, imagination, and hindered social interaction,2 while PDD refers to a group of conditions that include autistic disorder, Rett’s disorder, Asperger’s disorder, as well as a group of other related conditions.3 The first studies on autism were circulated in the 1960s, and many less severe types of autism were not identified until the 1980s. Since then, epidemiologists have conducted numerous surveys on autism that yielded higher prevalence rates year after year.4,5 This might be attributed to: increased awareness by both healthcare professionals and families of autistic children, and changes in diagnostic criteria. However, it is likely that the prevalence remains underestimated as many cases of autism are probably undiagnosed or unrecognized in the community, particularly the mild ones. This might be partly due to lack of awareness of both public and health care providers. Lack of screening programs and difficult access to care due to various reasons might have also contributed.6 Although there is little research into the global burden of autism, some studies in the USA and UK estimated the annual cost of autism on the economy and community to be more than several billion US dollars.7,8 The prevalence of autism is variable; Europe reported a median of 18.75 per 10,000, and the USA reported a median of 21.6 per 10,000. However, China reported a lower median of 11.6 per 10,000. Similarly, the male to female case ratio ranged from 1.33:1 to 16:1.1 Socioeconomical factors affect the prevalence rate prominently. For instance, studies in India have shown that most diagnosed cases belong to middle-class families. Upper class families do not frequent public health centers to treat autistic children, and families from low socioeconomic strata do not access such facilities unless the child is acutely ill.9-11 Obviously, the global prevalence for PDD was higher than that of autistic disorder due to the more inclusive definition of PDD.A multitude of heritable and non-heritable exposures were studied in relation to ASD.12 Based on extensive review of ASD epidemiology; Newschaffer et al12 suggested a model that categorized potential risk factors into: 1) genetic predisposition of the mother, 2) environmental factors acting the mother, 3) genetic predisposition of the child, and 4) environmental factors affecting the child. Early diagnosis and subsequent intervention for ASD and PDD are paramount, as research has shown the potential of greater benefit with early intervention.13-15The Gulf Cooperation Council (GCC) countries comprise 6 Arab countries, which are located in the Arab peninsula; namely: Bahrain, Kuwait, Qatar, Saudi Arabia, Sultanate of Oman, and United Arab Emirates (UAE). These countries have a shared geographical location, ethnic backgrounds, and life styles. Moreover, genetic exposures such as consanguinity and multiparity is common in this area. Epidemiological research into autism in the GCC is relatively new, and the burden of autism in this part of the world is still unclear. Our objective was to review the current state of knowledge of epidemiology on autism in the GCC, and make recommendations for future research.