Distribution of Hypoxia and Proliferation Associated Markers in Spontaneous Canine Tumors

The therapeutic response of malignant tumors depends on a number of factors associated with tumor microenvironments including the possibility that these microenvironments change during treatment. Two factors, tumor hypoxia and cell proliferation, have been examined in spontaneous canine tumors undergoing multifraction radiation therapy. The approach utilizes immunohistochemical analyses of hypoxia (CCI-103F) and proliferation associated (PCNA) antigens in biopsy samples taken before and after 5 daily fractions of 3 Gy (total dose 15 Gy). The tissue samples were formalin-fixed and paraffin-embedded for the immunohistochemical study. Immunostaining of the sections for PCNA and hypoxia marker reveals little or no overlap when the analysis is made prior to irradiation. An increased degree of overlap seems to occur after 15 Gy but the situation is complicated by a change towards more diffuse PCNA immunostaining in the cells of the irradiated tissues.     

Biochemical and molecular analyses in three patients with 3-hydroxy-3-methylglutaric aciduria

Two methods, spectrophotometry and HPLC, were compared in the analyses of 3-hydroxy-3-methylglutaryl-CoA lyase (HL) activity in three unrelated Czech patients with 3-hydroxy-3-methylglutaric (HMG) aciduria and their family members. The HL activities in cultured fibroblasts and/or isolated lymphocytes of probands were below the detection limits of the methods used. Both methods were also suitable for recognition of all heterozygotes in affected families. We searched for pathogenic mutations in the HL gene. Molecular analyses revealed that two patients are homozygous for known mutation H233R and R41Q, respectively, whereas the third patient is a compound heterozygote for the mutation H233R and a novel mutation Pro9fs(−1). This study expands the knowledge of the genotypic variability of the HMG aciduria. This revised version was published online in September 2006 with corrections to the Cover Date.     

Toward a national consensus: teaching radiobiology to radiation oncology residents

PURPOSE: The ASTRO Joint Working Group on Radiobiology Teaching, a committee composed of members having affiliations with several national radiation oncology and biology-related societies and organizations, commissioned a survey designed to address issues of manpower, curriculum standardization, and instructor feedback as they relate to resident training in radiation biology. METHODS AND MATERIALS: Radiation biology instructors at U.S. radiation oncology training programs were identified and asked to respond to a comprehensive electronic questionnaire dealing with instructor educational background, radiation biology course content, and sources of feedback with respect to curriculum planning and resident performance on standardized radiation biology examinations. RESULTS: Eighty-five radiation biology instructors were identified, representing 73 radiation oncology residency training programs. A total of 52 analyzable responses to the questionnaire were received, corresponding to a response rate of 61.2%. CONCLUSION: There is a decreasing supply of instructors qualified to teach classic, and to some extent, clinical, radiobiology to radiation oncology residents. Additionally, those instructors with classic training in radiobiology are less likely to be comfortable teaching cancer molecular biology or other topics in cancer biology. Thus, a gap exists in teaching the whole complement of cancer and radiobiology curricula, particularly in those programs in which the sole responsibility for teaching falls to one faculty member (50% of training programs are in this category). On average, the percentage of total teaching time devoted to classic radiobiology (50%), clinical radiobiology (30%), and molecular and cancer biology (20%) is appropriate, relative to the current makeup of the board examination. Nevertheless large variability exists between training programs with respect to the total number of contact hours per complete radiobiology course (ranging from approximately 10 to >50 h). A number of lecture topics, particularly in clinical radiobiology, are covered by fewer than 60% of training programs. A sizeable minority of radiation biology instructors are dissatisfied with the feedback they receive with respect to both course content and the performance of their residents on standardized radiobiology examinations administered by the American College of Radiology and/or the American Board of Radiology.     

Clenbuterol reduces degeneration of exercised or aged dystrophic (mdx) muscle

Evidence of dystrophic muscle degeneration in the hind limb muscles of young (20-week-old) treadmill-exercised or aged (87-week-old) sedentary mdx mice was greatly reduced by treatment with clenbuterol, a beta(2)-adrenoceptor agonist. Daily treadmill exercise for 10 weeks increased the size of regions within the mdx plantaris but not the soleus or gastrocnemius muscles, in which necrotic muscle fibers or the absence of fibers was observed. Clenbuterol reduced the size of these abnormal regions from 21% of total muscle cross-sectional area to levels (4%) found in sedentary mdx mice. In addition, the muscles obtained from aged clenbuterol-treated mdx or wild-type mice did not display the extensive fibrosis or fiber loss observed in untreated mdx mice. These observations are consistent with a mechanism of dystrophic muscle degeneration caused by work load-induced injury that is cumulative with aging and is opposed by beta(2)-adrenoceptor activation. Optimization of beta(2)-agonist treatment of muscular dystrophy in mdx mice may lead to a useful therapeutic modality for human forms of the disease.     

Clastogenic and aneugenic effects of tamoxifen and some of its analogues in hepatocytes from dosed rats and in human lymphoblastoid cells transfected with human P450 cDNAs (MCL-5 cells)

Tamoxifen and its analogues 4-hydroxytamoxifen, toremifene, 4- hydroxytoremifene, clomifene and droloxifene were tested for clastogenic effects in a human lymphoblastoid cell line (MCL-5) expressing elevated native CYP1A1 and containing transfected CYP1A2, CYP2A6, CYP2E1 and CYP3A4 and epoxide hydrolase and in a cell line containing only the viral vector (Ho1). MCL-5 or Ho1 cells were incubated with 4-hydroxytamoxifen, 4-hydroxytoremifene, clomifene or droloxifene and the incidence of micronuclei estimated. With MCL-5 cells there was an increase in micronuclei with 4-hydroxytamoxifen, 4- hydroxytoremifene and clomifene but not with droloxifene. With Ho1 cells only 4-hydroxytamoxifen and 4-hydroxytoremifene caused an increase in micronuclei. MCL-5 cells were incubated with tamoxifen, 4- hydroxytamoxifen, toremifene, droloxifene, clomifene or diethylstilbestrol (0.25-10 microg/ml) for 48 h and subjected to 3 h treatment with vinblastine (0.25 microg/ml) to arrest cells in metaphase. The incidence of cells with chromosomal numerical aberrations (aneuploidy) was increased in cells treated with tamoxifen, 4-hydroxytamoxifen, toremifene, clomifene and diethylstilbestrol but not droloxifene. The frequency of cells with structural abnormalities (excluding gaps) was increased in cells treated with tamoxifen and toremifene but not 4-hydroxytamoxifen, clomifene, droloxifene or diethylstilbestrol. The clastogenic activities of tamoxifen (35 mg/kg), toremifene (36.3 mg/kg), droloxifene (35.2 mg/kg) and diethylstilbestrol (25 mg/kg) were compared in groups of four female Wistar rats. Each chemical was dissolved in glycerol formal, administered as a single dose by gavage and hepatocytes isolated by collagenase perfusion 24 h later. The cells were cultured in the presence of epidermal growth factor (40 ng/ml) for 48 h, colchicine (10 microg/ml) being added for the final 3 h of incubation. At least 100 chromosomal spreads were examined from each animal for the presence of numerical and structural abnormalities. The incidences of aneuploidy following treatment were: tamoxifen 81%, toremifene 46%, droloxifene 9.6%, diethylstilbestrol 45.7%, vehicle control 5.3%. The incidences of chromosomal structural abnormalities excluding gaps were: tamoxifen 4.3%, toremifene 0.8%, droloxifene 0.5%, diethylstilbestrol 0.8%, control 0.5%. The incidence of chromosomal structural aberrations excluding gaps in the treated animals was not statistically significantly different from controls except in the tamoxifen-treated group. Tamoxifen (35 mg/kg per os) and toremifene (36.3 mg/kg per os) were dosed to rats for 4 weeks and chromosomal spreads made from hepatocytes. The incidences of aneuploidy were: tamoxifen 94%, toremifene 57%, control 6.5%. The incidences of chromosomal aberrations excluding gaps were: tamoxifen 12%, toremifene 1%, control 0.5%. The incidence of tamoxifen-induced chromosomal structural abnormalities was significantly elevated compared with control levels. The results demonstrate that tamoxifen and toremifene are the only two drugs tested in the study that cause chromosomal structural and numerical aberrations in vitro and tamoxifen is the only drug that induces both these effects in rat liver cells stimulated to divide in culture following oral dosing. Since chromosomal mutations require cell division for their manifestation and tamoxifen is the only compound of those tested that causes hyperplasia in the rat liver, chromosomal aberrations and aneuploidy in the rat liver would only be expected to occur following treatment with tamoxifen alone, although aneuploidy could be induced by toremifene in conjunction with a promoter such as phenobarbitone.      

Ki-ras mutations are an early event and correlate with tumor stage in transplacentally-induced murine lung tumors

A previous study from this laboratory demonstrated that treatment of pregnant mice with 3-methylcholanthrene (MC) caused lung tumors in the offspring at 1 year after birth, the incidence of which correlated with fetal inducibility of Cyp1a1. Analysis by PCR amplification and allele- specific hybridization (ASO) of paraffin-embedded tumors generated from that study revealed the presence of point mutations in exon 1 of the Ki- ras gene. This work has now been expanded by PCR amplification and ASO analysis of 31 additional lesions. Point mutations were found in 37 of the 47 (79%) lesions analyzed in this and the previous study, the majority of which were G-->T transversions in the first or second base of codon 12. The mutational spectrum appeared to be dependent on the relative stage of differentiation of the lesion, as both the incidence of mutation and type of mutation produced correlated with malignant progression. Mutations occurred in 60% of the hyperplasias, 80% of the adenomas and 100% of the adenocarcinomas. In the lesions with mutations, GLY12-->CYS12 transversions occurred in 100% of the hyperplasias, 42% of the adenomas and 14% of the adenocarcinomas. The GLY12-->VAL12 transversions occurred in none of the hyperplasias, 42% of the adenomas and 57% of the adenocarcinomas. The remaining mutations, which consisted of ASP12 transitions and ARG13 transversions, occurred only in adenomas (17%) and adenocarcinomas (29%). Between this study and our previous analyses, the identity of the mutations obtained by ASO were confirmed by sequence analysis of eight of the 37 lesions that harbored mutations at the Ki-ras gene locus. There were no differences in the type or incidence of mutations relative to the metabolic phenotype or sex of the mice. These data suggest that mutational activation of the Ki-ras gene locus is an early event in transplacental lung tumorigenesis, and that the type of mutations produced by exposure to chemical carcinogens can influence the carcinogenic potential of the tumor. This may have prognostic significance in determining the malignant progression of the neoplasm.      

Childhood acute lymphoblastic leukaemia presenting as jaundice

ABSTRACT An unusual presentation of acute lymphoblastic leukaemia (ALL) in a 6-year-old girl is reported. She presented with unilateral cervical lymphadenopathy, a mixed obstructive/cholestatic jaundice and a progressive pancytopenia. Ultrasound examination revealed an obstructed common bile duct with gross thickening of the wall of the duct and intrahepatic bile duct dilatation. The jaundice resolved with high dose intravenous (i.v.) methylprednisolone. It is postulated that this was due to infiltration of the common bile duct, given the failure to demonstrate any other cause for the bile duct pathology.