CAG and CTG repeat expansions are the cause of at least a dozen inherited neurological disorders. In these so-called “dynamic mutation” diseases, the expanded repeats display dramatic genetic instability, changing in size when transmitted through the germline and within somatic tissues. As the molecular basis of the repeat instability process remains poorly understood, modeling of repeat instability in model organisms has provided some insights into potentially involved factors, implicating especially replication and repair pathways. Studies in mice have also shown that the genomic context of the repeat sequence is required for CAG/CTG repeat instability in the case of spinocerebellar ataxia type 7 (SCA7), one of the most unstable of all CAG/CTG repeat disease loci. While most studies of repeat instability have taken a candidate gene approach, unbiased screens for factors involved in trinucleotide repeat instability have been lacking. We therefore attempted to use Drosophila melanogaster to model expanded CAG repeat instability by creating transgenic flies carrying trinucleotide repeat expansions, deriving flies with SCA7 CAG90 repeats in cDNA and genomic context. We found that SCA7 CAG90 repeats are stable in Drosophila, regardless of context. To screen for genes whose reduced function might destabilize expanded CAG repeat tracts in Drosophila, we crossed the SCA7 CAG90 repeat flies with various deficiency stocks, including lines lacking genes encoding the orthologues of flap endonuclease-1, PCNA, and MutS. In all cases, perfect repeat stability was preserved, suggesting that Drosophila may not be a suitable system for determining the molecular basis of SCA7 CAG repeat instability.

Current paradigms of peripheral B cell selection suggest that autoreactive B cells are controlled by clonal deletion, anergy, and developmental arrest. We report that changes to the human antibody repertoire likely resulting from these mechanisms both for a well-characterized autoreactivity from antibodies encoded by the V(H)4-34 gene and for other hallmarks of an autoreactive repertoire are apparent mainly for class-switched B cells and not for IgM germinal center, IgM memory, or IgM plasma cells. Other possible indicators of autoreactivity found selected with immunoglobulin class include J(H)6 gene segment usage, increased frequency of B cells with long third hypervariable regions, and distal J(kappa) gene segment bias. Of particular interest is the finding that B cells with these same characteristics are selected into the lineage of B cells that have undergone the unusual class switch from constant region C mu to C delta (C delta-CS). The C delta-CS population also displays an increased frequency of charged amino acids localized to the complementarity-determining regions, further suggesting autoreactivity, and evidence is presented that these B cells had undergone extensive receptor editing. Thus, the C delta-CS lineage may be a "sink" for B cells harboring autoreactive specificities in normal humans. A model for a new tolerizing mechanism that could account for the C delta-CS lineage is presented.

To identify genes expressed in interesting spatial and temporal patterns during development, we mobilized transposons carrying the yeast transcriptional activator, GAL4, to create new insertions throughout the Drosophila genome. At these new sites, neighboring enhancers drive expression of GAL4 in a pattern similar to that of nearby genes (Brand and Perrimon, 1993). Since GAL4 binds to a UAS target sequence, flies containing a GAL4 transposon can activate transcription of a UAS-linked gene, thereby resulting in controlled expression of that secondary gene. Thus, the GAL4 P element is a useful tool both for identifying genes of interest and for ectopically expressing genes in novel tissues or at specific developmental stages. We mobilized a GAL4 insertion on the second chromosome (P{GawB} CY2; Queenan et al., 1997) to generate a collection of GAL4-expressing enhancer trap lines. F1 “jumpstarter” males (w/Y; GAL4/CyO; Sb 2-3/ ) were mated in vials to attached-X (C(1)DX/Y) females. From 200 such crosses, red-eyed, curly-winged, long-bristled sons (bearing a new GAL4 insertion) were selected and mated individually to w/w females to establish 50 lines. Chromosome-mapping data, enhancer trap pattern, and other phenotypic characters demonstrated that four insertions were duplicates, yielding a total of 46 independent lines (Table 1). All X and third chromosome lines are viable, although three lines exhibit reduced viability or fertility (lines 15, 45, and 50). We could not determine the viability of the CyOlinked lines since the balancer chromosome is homozygous lethal. The genotypes of the resulting stocks are: X-chromosome lines, w P{GawB}, second-chromosome lines, y w; Pin/CyO, P{GawB}, and third chromosome lines, y w; III P{GawB}. Line 15 also carries the FM6, y w Bar balancer while lines 45 and 50 contain TM3, Sb Ser. Finally, line number three exhibits a striking eye phenotype consisting of a variable loss of eye pigment in a gradient along the anterior/posterior axis. Surprisingly, this line lacks detectable GAL4 expression in larval eye discs. We characterized sequences flanking a subset of the insertions in our collection by the inverse PCR method (http:www.fruitfly.org/methods/). Table 1 lists neighboring predicted or known genes. To identify and visualize the morphology of cells expressing GAL4, we crossed females from each line to males carrying UAS-taulacZ, which links the -galactosidase enzyme to the tubulin-binding protein Tau (Hildago et al., 1995). Some lines (1, 7, 10 (males only), 14, 22, 25, 26, 32, 35, 36, and 41) were lethal in pupal stages when crossed to this reporter; therefore, to assay ovary and testis expression, we crossed these strains to UAS-lacZ.NZ, which contains a nuclear-localization signal (Y. Hiromi and S. West, unpubl. results). Tables 1 and 2 describe the expression patterns for each line and Figure 1 shows selected patterns that illustrate key points. More detailed descriptions of all the patterns are available upon request. All lines exhibit interesting staining in at least one developmental stage. As commonly noted, we observe patchy or variable GAL4 expression in many lines. For example, GAL4 drives embryonic PNS expression in 18 lines (Table 1). While all embryos within a given strain exhibit a general PNS pattern that is consistent, within an individual embryo, slight variations often occur between segmental repeats (e.g., brackets in Fig. 1a). Only a few lines express GAL4 in a single tissue at a specific developmental stage. For example, line 20 drives expression in the longitudinal visceral mesoderm from stage 12 onwards (Fig. 1c). When crossed to UASGFP, line 20 can be used to visualize this highly migratory tissue in vivo. As Tables 1 and 2 indicate, many lines express GAL4 in identical tissues. Nevertheless, important differences may exist in the distribution of expression within each tissue. For example, Fig. 1f–i shows representative wing discs from four lines that express GAL4 in all imaginal discs, the brain, and the central nervous system (IGD, brain, and CNS). The distinct patterns shown in the wing discs presumably reveal specific regulatory elements that govern expression of nearby

Protein kinase A (PKA) holoenzyme is anchored to specific subcellular regions by interactions between regulatory subunits (Pka-R) and A-kinase anchoring proteins (AKAPs). We examine the functional importance of PKA anchoring during Drosophila oogenesis by analyzing membrane integrity and actin structures in mutants with disruptions in Akap200, an AKAP. In wild-type ovaries, Pka-RII and Akap200 localized to membranes and to the outer rim of ring canals, actin-rich structures that connect germline cells. In Akap200 mutant ovaries, Pka-RII membrane localization decreased, leading to a destabilization of membrane structures and the formation of binucleate nurse cells. Defects in membrane integrity could be mimicked by expressing a constitutively active PKA catalytic subunit (Pka-C) throughout germline cells. Unexpectedly, nurse cells in Akap200 mutant ovaries also had enlarged, thin ring canals. In contrast, overexpressing Akap200 in the germline resulted in thicker, smaller ring canals. To investigate the role of Akap200 in regulating ring canal growth, we examined genetic interactions with other genes that are known to regulate ring canal morphology. Akap200 mutations suppressed the small ring canal phenotype produced by Src64B mutants, linking Akap200 with the non-receptor tyrosine kinase pathway. Together, these results provide the first evidence that PKA localization is required for morphogenesis of actin structures in an intact organism.

Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative disorder caused by the expansion of a CAG repeat encoding a polyglutamine tract in ataxin-2, the SCA2 gene product. The normal cellular function of ataxin-2 and the mechanism by which polyglutamine expansion of ataxin-2 causes neurodegeneration remain unknown. In this study we have used genetic and molecular approaches to investigate the function of a Drosophila homolog of the SCA2 gene (Datx2). Like human ataxin-2, Datx2 is found throughout development in a variety of tissue types and localizes to the cytoplasm. Mutations that reduce Datx2 activity or transgenic overexpression of Datx2 result in female sterility, aberrant sensory bristle morphology, loss or degeneration of tissues, and lethality. These phenotypes appear to result from actin filament formation defects occurring downstream of actin synthesis. Further studies demonstrate that Datx2 does not assemble with actin filaments, suggesting that the role of Datx2 in actin filament formation is indirect. These results indicate that Datx2 is a dosage-sensitive regulator of actin filament formation. Given that loss of cytoskeleton-dependent dendritic structure defines an early event in SCA2 pathogenesis, our findings suggest the possibility that dysregulation of actin cytoskeletal structure resulting from altered ataxin-2 activity is responsible for neurodegeneration in SCA2.

Here we describe our analyses of Rhino, a novel member of the Heterochromatin Protein 1(HP1) subfamily of chromo box proteins. rhino (rhi) is expressed only in females and chiefly in the germline, thus providing a new tool to dissect the role of chromo-domain proteins in development. Mutations in rhi disrupt eggshell and embryonic patterning and arrest nurse cell nuclei during a stage-specific reorganization of their polyploid chromosomes, a mitotic-like state called the "five-blob" stage. These visible alterations in chromosome structure do not affect polarity by altering transcription of key patterning genes. Expression levels of gurken (grk), oskar (osk), bicoid (bcd), and decapentaplegic (dpp) transcripts are normal, with a slight delay in the appearance of bcd and dpp mRNAs. Mislocalization of grk and osk transcripts, however, suggests a defect in the microtubule reorganization that occurs during the middle stages of oogenesis and determines axial polarity. This defect likely results from aberrant Grk/Egfr signaling at earlier stages, since rhi mutations delay synthesis of Grk protein in germaria and early egg chambers. In addition, Grk protein accumulates in large, actin-caged vesicles near the endoplasmic reticulum of stages 6-10 egg chambers. We propose two hypotheses to explain these results. First, Rhi may play dual roles in oogenesis, independently regulating chromosome compaction in nurse cells at the end of the unique endoreplication cycle 5 and repressing transcription of genes that inhibit Grk synthesis. Thus, loss-of-function mutations arrest nurse cell chromosome reorganization at the five-blob stage and delay production or processing of Grk protein, leading to axial patterning defects. Second, Rhi may regulate chromosome compaction in both nurse cells and oocyte. Loss-of-function mutations block nurse cell nuclear transitions at the five-blob stage and activate checkpoint controls in the oocyte that arrest Grk synthesis and/or inhibit cytoskeletal functions. These functions may involve direct binding of Rhi to chromosomes or may involve indirect effects on pathways controlling these processes.

Seventy patients with T3N0 glottic cancer who received a total dose of 60 Gy in twenty-five 2.4 Gy daily fractions over 5 weeks had an initial post radiation primary recurrence free rate of 65% at 5 years. This provides one more point for the dose response for T3 glottic cancer Radiother Oncol (1999) 15.

Purpose: To examine the effect of the time interval (interval) between breast-conserving surgery (BCS) and the start of radiation therapy (RT) on the subsequent risk of ipsilateral breast cancer recurrence (IBR),Methods and Materials: We reviewed interval and a number of prognostic and treatment factors among 1,962 women treated with BCS and RT for invasive breast cancer diagnosed between January 1, 1989 and December 31, 1993 in British Columbia, Canada. Subjects were female, less than 90 years old at diagnosis, not treated with chemotherapy, not stage T4 or M1, and had survived more than 30 days from diagnosis. The cumulative incidence of IBR was estimated in four interval groups: 0-5, 6-8, 9-12, and 13+ weeks. Only 23 women had an interval of greater than 20 weeks between BCS and start of RT, To assess whether an imbalance of prognostic and treatment factors could be obscuring real differences between the interval groups, Cox proportional hazards regression analyses were conducted,Results: Median follow-up was 71 months. The crude incidence of IBR for the entire sample was 3.9%. The cumulative incidence of IBR in the 6-8, 9-12, and 13+ week groups was not statistically significantly different from the cumulative incidence of IBR in the 0-5 week group. Multivariate analyses demonstrated that patients not using tamoxifen (p = 0.027) and those with grade 3 histology (p = 0.003) were more likely to recur in the breast. Interval between BCS and RT was not a statistically significant predictor of breast recurrence when entered into a model incorporating tamoxifen use and tumor grade (0-5 vs, 6-8 weeks, p = 0.872; 0-5 vs. 9-12 weeks, p = 0.665; 0-5 vs. 13+ weeks, p = 0.573).Conclusions: We found no univariate or multivariate difference in ipsilateral breast cancer recurrence between intervals of 0 to 20 weeks from breast conserving surgery to start of radiation therapy, in a population-based, low risk group of women not receiving adjuvant chemotherapy, after controlling for other factors important in predicting ipsilateral breast cancer recurrence. (C) 2000 Elsevier Science Inc.

Since 1979, consensus guidelines have been produced for radiation treatment practice in British Columbia. These guidelines have been revised, updated and circulated to all radiation oncologists periodically. A comprehensive computer database for all patients receiving radiation treatment in the province was established in 1984. Between 1985 and 1996 inclusive, 7667 prostate cancer patients and 9748 breast cancer patients received primary radical radiation treatment or adjuvant postsurgical treatment. Palliative treatments and the treatment of other disease sites are not included in this review. Compliance of these treatments with the published guidelines is reported. Over 98% of patients completed treatment as planned and, in the last 3 years, more than 90% of patients received a guideline dose to either the prostate or the breast. Compliance was less in the treatment of the regional lymph nodes in breast cancer. Practice in prostate cancer tended to precede the changes in the guidelines. This was not the case for breast cancer.

The comet assay is a single-cell gel electrophoresis technique that measures DNA damage in individual cells. Since radiation produces 3-4 times more DNA damage in well-oxygenated cells compared with hypoxic cells, this assay can quantify the fraction of radiation-resistant hypoxic cells found in many solid tumours. This paper summarizes our results with 73 accessible metastatic tumours irradiated with palliative intent. Hypoxic fractions ranged from 0.0 to 0.67 with a mean of 0.15; 62% of these advanced tumours showed a hypoxic fraction > 0.05. Comparisons between two sequential aspirates in 33 tumours gave a slope of 0.92 (r2 = 0.88), suggesting that a single aspirate is generally representative of the tumour. A limitation, however, is that the hypoxic fraction could not be measured in clinical samples given a conventional dose of 2 Gy.

Background and purpose: The use of ipsilateral irradiation techniques to treat patients with carcinoma of the tonsil reduces the acute radiation reaction in the contralateral pharynx and late damage to the contralateral salivary tissue. However, this may also spare microscopic disease in apparently uninvolved contralateral lymph nodes. The purpose of this study was to analyse the survival and recurrence rates and sites of recurrance in a group of patients with carcinoma of the tonsil treated with ipsilateral techniques.Materials and methods: Between 1975 and 1993, 271 patients with invasive squamous cell cancer of the tonsil were referred to the Vancouver Cancer Centre (VCC). One hundred and seventy-eight received ipsilateral radiation treatment. Three received surgery only, six post-operative radiation, 12 supportive treatment only and 72 bilateral radiation treatment. In the absence of bilateral neck nodes and extensive lymphodenopathy, field sizes were generally kept small to include the primary tumour and the first echelon of nodes. The most common dose was 60 Gy in 25 daily fractions in 5 weeks (2.4 Gy per day).Results and discussion: The disease specific survival for all patients treated by radical radiation treatment was 61% at 5 years, For the 178 patients who received ipsilateral radiation treatment the overall primary tumour control rate by ipsilateral radiation treatment alone was 75% and for T1 and T2 tumours 84%. Eight (7.5%) of 101 of these patients with NO nodes at presentation and without prior failure at the primary site, developed nodal recurrence (four within the initially radiated high dose volume), Two developed contralateral nodes, and two developed field edge nodal recurrence, one cured by surgery. In 54 patients with N1 disease, five developed nodal recurrence, two within field, two contralateral, one of whom was cured by surgery, and one at field edge. In 23 patients with N2a, N2b or N3 disease node control was achieved from radiation treatment in 11 and two more were cured by surgery. All nodal failures were within the radiated volume. Overall, 10 of the 25 patients with nodal failure were cured by subsequent surgery.Conclusions: Ipsilateral treatment of patients with carcinoma of the tonsil gives survival results that are at least as good as those reported with bilateral treatment with fewer side effects and a very low risk of failure in the contralateral neck, (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.

The cut gene of Drosophila melanogaster encodes a homeodomain protein that regulates a soma-to-germline signaling pathway required for proper morphology of germline cells during oogenesis. cut is required solely in somatic follicle cells, and when cut function is disrupted, membranes separating adjacent nurse cells break down and the structural integrity of the actin cytoskeleton is compromised. To understand the mechanism by which cut expression influences germline cell morphology, we determined whether binucleate cells form by defective cytokinesis or by fusion of adjacent cells. Egg chambers produced by cut, cappuccino, and chickadee mutants contained binucleate cells in which ring canal remnants stained with antibodies against Hu-li tai shao and Kelch, two proteins that are added to ring canals after cytokinesis is complete. In addition, defects in egg chamber morphology were observed only in middle to late stages of oogenesis, suggesting that germline cell cytokineses were normal in these mutants. cut exhibited dose-sensitive genetic interactions with cappuccino but not with chickadee or other genes that regulate cytoskeletal function, including armadillo, spaghetti squash, quail, spire, Src64B, and Tec29A. Genomic regions containing genes that cooperate with cut were identified by performing a second-site noncomplementing screen using a collection of chromosomal deficiencies. Sixteen regions that interact with cut during oogenesis and eight regions that interact during the development of other tissues were identified. Genetic interactions between cut and the ovarian tumor gene were identified as a result of the screen. In addition, the gene agnostic was found to be required during oogenesis, and genetic interactions between cut and agnostic were revealed. These results demonstrate that a signaling pathway regulating the morphology of germline cells is sensitive to genetic doses of cut and the genes cappuccino, ovarian tumor, and agnostic. Since these genes regulate cytoskeletal function and cAMP metabolism, the cut-mediated pathway functionally links these elements to preserve the cytoarchitecture of the germline cells.

Background and purpose: To study the effect of accelerated radiation treatment in locally advanced head and neck cancer, a prospective randomised trial was conducted comparing two fractionation schemes which differed only in the overall treatment time; fraction size and total dose were the same in both arms.Materials and methods: Eighty-two patients with stage 3 or 4 squamous cell cancer of the oral cavity, oropharynx, hypopharynx or larynx considered suitable for radical radiotherapy as primary treatment were stratified by site and stage and randomised to receive 66 Gy in 33 2 Gy daily fractions in 45-48 days (o.d.) or 66 Gy in 33 2 Gy twice daily fractions in 22-25 days (b.i.d.).Results: An initial improved clinical response in tumour control in the b.i.d. arm was not sustained and the 3 year recurrence free survival and overall survival rates were similar and not significantly different. The actuarial recurrence free survival was 49.1% in the b.i.d. arm and 44.3% in the o.d. arm. The disease free 3 year survival was 59.4% and 56.8%, respectively. The acute and late normal tissue effects were scored using the RTOG scale. As expected the acute effects were much greater in the b.i.d. arm. The combined grade 3 and 4 late effects were similar but the proportion of grade 4 reactions was significantly higher in the b.i.d. arm. The increase in grade 4 toxicity caused the trial to be discontinued after 82 of the planned 226 patients had been randomised.Conclusions: The severity of the normal tissue effects and a failure to demonstrate sustained local control does not support this frationation scheme in patients with such extensive head and neck cancer. (C) 1997 Elsevier Science Ireland Ltd.

Purpose: To illustrate some of the radiation treatment techniques with asymmetric collimators in one field dimension.Methods and Materials: Treatment planning for various sites is done with an in-house developed treatment planning system. Dose distributions in the central plane are illustrated.Results: The use of asymmetric collimation, in addition to being a replacement for cerrobend and lead blocks, can facilitate treatment setup with boost fields and with half-beam asymmetric fields as in matching two adjacent fields, in avoiding nearby critical organ or tissue, and in tangential breast treatment. The use of asymmetric collimators would alter the dose distribution across the radiation field and should be accounted for during treatment planning. In conjunction with arc rotation or multiple asymmetric fields, two-dimensional conformal radiotherapy is possible.Conclusion: The full potential of asymmetric collimation requires the use of a proper treatment planning algorithm. Some of the treatment techniques with asymmetric collimation in one field dimension are shown here. (C) 1997 Elsevier Science Inc.

Communications between the germline and the soma during Drosophila oogenesis have been previously shown to be essential for the formation of egg chambers and to establish polarity in the developing oocyte. In this report, we demonstrate that the function of a somatically expressed gene, cut, is critical for maintaining the structural integrity of germline-derived cells and their arrangement within an egg chamber. Genetic manipulations of cut activity resulted in defective packaging of germline-derived cysts into egg chambers and disintegration of the structural organization of oocyte-nurse cell complexes to generate multinucleate germline-derived cells. We also found that cut interacts genetically with the Notch gene and with the catalytic subunit of Protein kinase A gene during egg chamber morphogenesis. Since cut expression is restricted to the somatic follicle cells and cut mutant germline clones are phenotypically normal, we propose that the defects in the assembly of egg chambers and the changes in germline cell morphology observed in cut mutant egg chambers are the result of altered interactions between follicle cells and germline cells. cut encodes a nuclear protein containing DNA-binding motifs, and we suggest that it participates in intercellular communications by regulating the expression of molecules that directly participate in this process.

The alkaline comet assay has previously been used to estimate the fraction of radiobiologically hypoxic cells in tumours from patients undergoing palliative radiotherapy for advanced breast and head and neck cancer. Results obtained from fine needle aspirate biopsies (FNABs) using this method indicate considerable heterogeneity in hypoxic fraction between tumours. Heterogeneity between 3 aspirates taken from the same 10 tumours immediately following single doses of 3.5 to 5 Gy is now examined. Results indicate that a single fine needle aspirate is reasonably representative for DNA damage and DNA content. However, difficulties were encountered in obtaining an adequate sample of tumour cells after the final radiation treatment. The average hypoxic fraction decreased from 14% after the first dose to 9% after the last dose, and in 3 tumours which could be evaluated after both the first and last fraction, the hypoxic fraction decreased in two but increased in the third. Rejoining of DNA strand breaks was observed between sequential aspirates indicating that pooling of samples for analysis may not be advisable using this method.

Background and purpose: Acetylsalicylic acid (ASA) can reduce the incidence of stroke and myocardial infarction by inhibiting platelet-fibrin thrombi in small blood vessels. To determine if ASA could reduce late effects of radiation therapy mediated by damage to small blood vessels, a prospective, placebo-controlled, double-blind trial was conducted in women with early breast cancer, receiving radiotherapy to the conserved breast.Materials and methods: Cosmetic outcome and late radiotherapy effects were recorded prospectively for 186 women with TI or T2, pathologically node-negative breast cancer treated with breast conservation and randomized to receive ASA (325 mg daily) or placebo for I year from the start of radiation therapy. Radiation was a tangent pair to the breast alone delivering a modal dose of 44 Gy in 16 daily fractions in 22-25 days.Results: Median follow-up is 6.5 years. The use of ASA has not had any effect on the acute (erythema, edema or discomfort) or late (induration, telangiectasia) effects of radiotherapy (all P>0.10), the patients' or physicians' assessment of the cosmetic outcome (all P>0.25) or rates of breast recurrence (P>0.25).Conclusion: ASA cannot be recommended to improve the outcome of radiotherapy complementing breast conserving surgery.