Adaptive radiotherapy for breast cancer.

Research in the field of local and locoregional breast cancer radiotherapy aims to maintain excellent oncological outcomes while reducing treatment-related toxicity. Adaptive radiotherapy (ART) considers variations in target and organs at risk (OARs) anatomy occurring during the treatment course and integrates these in re-optimized treatment plans. Exploiting ART routinely in clinic may result in smaller target volumes and better OAR sparing, which may lead to reduction of acute as well as late toxicities. In this review MR-guided and CT-guided ART for breast cancer patients according to different clinical scenarios (neoadjuvant and adjuvant partial breast irradiation, whole breast, chest wall and regional nodal irradiation) are reviewed and their advantages as well as challenging aspects discussed.

Synthetic CT for single-fraction neoadjuvant partial breast irradiation on an MRI-linac.

A synthetic computed tomography (sCT) is required for daily plan optimization on an MRI-linac. Yet, only limited information is available on the accuracy of dose calculations on sCT for breast radiotherapy. This work aimed to (1) evaluate dosimetric accuracy of treatment plans for single-fraction neoadjuvant partial breast irradiation (PBI) on a 1.5 T MRI-linac calculated on a) bulk-density sCT mimicking the current MRI-linac workflow and b) deep learning-generated sCT, and (2) investigate the number of bulk-density levels required. For ten breast cancer patients we created three bulk-density sCTs of increasing complexity from the planning-CT, using bulk-density for: (1) body, lungs, and GTV (sCTBD1); (2) volumes for sCTBD1plus chest wall and ipsilateral breast (sCTBD2); (3) volumes for sCTBD2plus ribs (sCTBD3); and a deep learning-generated sCT (sCTDL) from a 1.5 T MRI in supine position. Single-fraction neoadjuvant PBI treatment plans for a 1.5 T MRI-linac were optimized on each sCT and recalculated on the planning-CT. Image evaluation was performed by assessing mean absolute error (MAE) and mean error (ME) in Hounsfield Units (HU) between the sCTs and the planning-CT. Dosimetric evaluation was performed by assessing dose differences, gamma pass rates, and dose-volume histogram (DVH) differences. The following results were obtained (median across patients for sCTBD1/sCTBD2/sCTBD3/sCTDLrespectively): MAE inside the body contour was 106/104/104/75 HU and ME was 8/9/6/28 HU, mean dose difference in the PTVGTVwas 0.15/0.00/0.00/-0.07 Gy, median gamma pass rate (2%/2 mm, 10% dose threshold) was 98.9/98.9/98.7/99.4%, and differences in DVH parameters were well below 2% for all structures except for the skin in the sCTDL. Accurate dose calculations for single-fraction neoadjuvant PBI on an MRI-linac could be performed on both bulk-density and deep learning sCT, facilitating further implementation of MRI-guided radiotherapy for breast cancer. Balancing simplicity and accuracy, sCTBD2showed the optimal number of bulk-density levels for a bulk-density approach.

Identification of genes targeted by FSH and oocytes in porcine granulosa cells.

In the mammalian ovarian follicle maturing oocytes are nurtured and supported by surrounding somatic cells, the mural granulosa cells and the cumulus cells. These cells are regulated by follicle-stimulating hormone (FSH), originating from the pituitary, and paracrine factors derived from the oocyte. To gain insight into the mechanisms involved in the regulation of granulosa cell function, this study aimed to identify genes in mural granulosa cells that are regulated by FSH and oocyte secreted factors using the pig as a model organism. Mural granulosa cells were collected from 3-6 mm follicles from sow ovaries and cultured in serum free medium in the presence or absence of FSH and/or isolated cumulus oocyte complexes (COCs). FSH significantly increased both the metabolic activity and progesterone production of granulosa cells, while the presence of COCs reversed these FSH effects. Expression levels of mRNA in the absence/presence of FSH and COCs were analyzed on porcine specific microarrays representing 11,300 genes. Both previously identified and novel FSH target genes as well as some oocyte affected genes were found. Expression of inhibitor of DNA binding protein 2 and 3, ID2 and ID3, was decreased by FSH but increased by COCs, as validated by quantitative PCR. These proteins function as dominant negative basic helix loop helix (bHLH) transcription factors and since all regulated genes contain the consensus E-box sequence that can bind bHLH factors, our data suggest that FSH and COCs may regulate granulosa cell function by tuning the activity of bHLH factors, through ID2 and ID3.

High-resolution genetic mapping of mammalian motor activity levels in mice.

The generation of motor activity levels is under tight neural control to execute essential behaviors, such as movement toward food or for social interaction. To identify novel neurobiological mechanisms underlying motor activity levels, we studied a panel of chromosome substitution (CS) strains derived from mice with high (C57BL/6J strain) or low motor activity levels (A/J strain) using automated home cage behavioral registration. In this study, we genetically mapped the expression of baseline motor activity levels (horizontal distance moved) to mouse chromosome 1. Further genetic mapping of this trait revealed an 8.3-Mb quantitative trait locus (QTL) interval. This locus is distinct from the QTL interval for open-field anxiety-related motor behavior on this chromosome. By data mining, an existing phenotypic and genotypic data set of 2445 genetically heterogeneous mice (http://gscan.well.ox.ac.uk/), we confirmed linkage to the peak marker at 79 970 253 bp and refined the QTL to a 312-kb interval containing a single gene (A830043J08Rik). Sequence analysis showed a nucleotide deletion in the 3' untranslated region of the Riken gene. Genome-wide microarray gene expression profiling in brains of discordant F(2) individuals from CS strain 1 showed a significant upregulation of Epha4 in low-active F(2) individuals. Inclusion of a genetic marker for Epha4 confirmed that this gene is located outside of the QTL interval. Both Epha4 and A830043J08Rik are expressed in brain motor circuits, and similar to Epha4 mutants, we found linkage between reduced motor neurons number and A/J chromosome 1. Our findings provide a novel QTL and a potential downstream target underlying motor circuitry development and the expression of physical activity levels.

cDNA microarray profiles of canine mammary tumour cell lines reveal deregulated pathways pertaining to their phenotype.

Mammary cancer is the most common type of cancer in female dogs with a lifetime risk of over 24% when dogs are not spayed. The elucidation of the complete canine genome opens new areas for development of cancer therapies. These should be tested first by in vitro models such as cell lines. However, to date, no canine mammary cell lines have been characterized by expression profiling. In this study, canine mammary tumour cell lines with histologically distinct primary tumours of origin were characterized using a newly developed canine cDNA microarray. Comparisons of gene expression profiles showed enrichment for distinct biological pathways and were related to biological properties of the cell lines such as growth rate and in vitro tumourigenicity. Additionally, gene expression profiles of cell lines also showed correspondence to their tumour of origin. Major differences were found in Wnt, cell cycle, cytokine/Rho-GTPase, alternative complement and integrin signalling pathways. Because these pathways show an overlap at the molecular level with those found in human breast cancer, the expression profiling of spontaneous canine mammary cancer may also function as a biological sieve to identify conserved gene expression or pathway profiles of evolutionary significance that are involved in tumourigenesis. These results are the basis for further characterization of canine mammary carcinomas and development of new therapies directed towards specific pathways. In addition these cell lines can be used to further investigate identified deregulated pathways and characterize until now unannotated genes.

Changes in mRNA expression profile underlie phenotypic adaptations in creatine kinase-deficient muscles.

We have studied the mechanisms that regulate the remodeling of the glycolytic, mitochondrial and structural network of muscles of creatine kinase M (M-CK)/sarcomeric mitochondrial creatine kinase (ScCKmit) knockout mice by comparison of wild-type and mutant mRNA profiles on cDNA arrays. The magnitudes of changes in mRNA levels were most prominent in M-CK/ScCKmit (CK(-/-)) double mutants but did never exceed those of previously observed changes in protein level for any protein examined. In gastrocnemius of CK(-/-) mice we measured a 2.5-fold increase in mRNA level for mitochondrial encoded cytochrome c oxidase (COX)-III which corresponds to the increase in protein content. The level of the nuclear encoded mRNAs for COX-IV, H(+)-ATP synthase-C, adenine nucleotide translocator-1 and insulin-regulatable glucose transporter-4 showed a 1.5-fold increase, also in agreement with protein data. In contrast, no concomitant up-regulation in mRNA and protein content was detected for the mitochondrial inorganic phosphate-carrier, voltage-dependent anion channel and certain glycolytic enzymes. Our results reveal that regulation of transcript level plays an important role, but it is not the only principle involved in the remodeling of mitochondrial and cytosolic design of CK(-/-) muscles.

Interlocked circle formation by group I introns: structural requirements and mechanism.

Precursor RNA transcribed from the yeast mitochondrial gene coding for the large ribosomal RNA contains a group I intron that can excise itself in vitro. Apart from group I specific sequence elements the intron also contains a gene encoding a DNA endonuclease involved in intron dispersal. A precursor RNA derivative from which this gene has been removed self-splices efficiently, but due to activation of cryptic opening sites located in the 5' exon, the 3' part of this exon is sometimes co-excised with the intron. Upon further reaction, this enlarged intron molecules give rise to interlocked circles, comprising small circles derived from 5' exon parts and large circles of the intron. Sequence comparison between cryptic opening sites and authentic splice sites reveals in most cases homology with the 3' exon part that is capable of interacting with the Internal Guide Sequence. The role of the IGS was further substantiated by replacing the cryptic opening sites with well defined sequences of authentic splice sites: one corresponding to the 3' splice site and its mutant derivatives, the other to a fragment containing the natural 5'-3' exon junction. Precursor RNAs derived from these constructs give rise to interlocked circles, and mutation studies confirm that the 3' exon nucleotides flanking a 3' splice site are essential for their formation. The results underline the crucial role of the IGS in interlocked circle formation which behaves similarly as in the normal self-splicing reactions. It has been proposed that the two short helices formed by basepairing of the IGS with the 5' and 3' exon can co-axially stack on top of each other forming a quasi continuous RNA double helix or pseudoknot. We present a model explaining how transesterification reactions of a mutant precursor RNA in such a pseudoknot can lead to interlocked circles. The experiments support the notion that a similar structure is also operative in splicing of wild type precursor RNA.

Splice site selection by intron aI3 of the COX1 gene from Saccharomyces cerevisiae.

Interactions of the 5' and 3' splice sites with intron internal sequences of the yeast mitochondrial group I intron aI3 were studied using mutation analysis. The results can be fully explained by the splice guide model in which the splice sites are defined by the Internal Guide Sequence. No evidence was found for an alternative interaction between intron nucleotides preceding the 3' splice site and nucleotides in the vicinity of the core region as was found for the Tetrahymena intron. Our results also suggest that binding of the 5' and 3' splice site nucleotides to the IGS can not take place simultaneously. The intron must therefore undergo conformational changes as the reaction proceeds from the first step of self splicing, GTP attack at the 5' splice site, to exon ligation, the second step.

The mechanism of group I self-splicing: an internal guide sequence can be provided in trans.

We have reconstituted a group I self-splicing reaction between two RNA molecules with different functional RNA parts: a substrate molecule containing the 5' splice site and a functional internal guide sequence (IGS), and a ribozyme molecule with core structure elements and splice sites but a mutated IGS. The 5' exon of the substrate molecule is ligated in trans to the 3' exon of the ribozyme molecule, suggesting that the deficient IGS in the ribozyme can be replaced by an externally added IGS present on the substrate molecule. This result is different from catalysis mediated by proteins where it is not possible to dissect the specificity of an enzyme from its catalytic activity.

Mitochondrial RNA polymerase of Saccharomyces cerevisiae: composition and mechanism of promoter recognition.

Mitochondrial RNA polymerase of Saccharomyces cerevisiae consists of two different proteins: a core RNA polymerase of 145 kd and a specificity factor of 43 kd, which contributes the capacity to recognize promoters of the various genes encoded in the mitochondrial genome. We purified both components by SDS-PAGE, followed by renaturation to the active state. The two components were used either singly or in combination to study their interactions with promoter-containing DNA fragments. The core component showed random and weak interaction with DNA, the specificity factor none at all, whereas both components together specifically bound to a promoter. In DNase I footprinting experiments, promoter-bound RNA polymerase protected a short region of DNA flanked by hypersensitivity sites and centred around the position at which RNA synthesis starts. The initial phase of transcription gave rise to specific changes in this footprint: the upstream border remained at the same position up to synthesis of a 4-nt RNA chain, whereas at the downstream border progressive disappearance of hypersensitivity sites took place.

RNA polymerase induces DNA bending at yeast mitochondrial promoters.

Yeast mitochondrial RNA polymerase can bind specifically to promoter-containing DNA fragments in vitro as detected by DNAse I or methidiumpropyl-EDTA. Fe(II) protection assays and gel retardation experiments. Retardation of RNA polymerase-DNA complexes was most pronounced when the promoter was located in the middle of a DNA fragment and was diminished when RNA polymerase was bound near one of the ends. This indicates that upon RNA polymerase-binding the DNA undergoes a conformational change which is most likely a bend. The degree of introduced bending correlated with the efficiency of transcription and promoter-binding in a series of promoter mutants, suggesting that bending is a functional event during promoter utilisation.

Discrimination between RNA circles, interlocked RNA circles and lariats using two-dimensional polyacrylamide gel electrophoresis.

Two-dimensional polyacrylamide gel electrophoresis can be used to identify structural forms of RNA such as linear RNA, circular RNA, interlocked circles and lariats. The procedure is based upon the characteristic migration behaviour of the degradation products derived from the intact structures present already before the start of the experiment or formed during or after electrophoresis in the first dimension. After autoradiography to detect the positions of the radiolabeled RNA molecules, circles broken during electrophoresis of the first dimension give rise to horizontal lines touching the diagonal formed by linear RNAs at a point corresponding to the length of the RNA circle from which it was derived. Products derived from interlocked RNA circles by breakage after completion of the first dimension appear on a vertical line underneath the intact complex and consist of free RNA circles and their linear derivatives. Broken lariats give rise to two lines depending on the location of the break. Lariats with broken tails are present on a line to a position that corresponds to the length of their tail and that runs parallel to the diagonal formed by linear products. Lariats with a broken eye form a line running from the position of the intact product to the diagonal formed by the linear RNAs.

Effect of point mutations on in vitro transcription from the promoter for the large ribosomal RNA gene of yeast mitochondria.

Initiation of transcription on mitochondrial DNA of Saccharomyces cerevisiae was studied in an in vitro system with a mtRNA polymerase fraction reconstituted from separately purified components and with DNA templates containing the promoter of the gene coding for large rRNA. The effect of various point mutations in this promoter region was quantitated in assays containing a wildtype promoter in equimolar amount as internal control. Despite the strong conservation around the position at which RNA initiation occurs (ATATAAGTApuTA, initiation nucleotide underlined), none of the single point mutations abolished transcription-initiation completely. Some reduce the efficiency of initiation to 10-20% compared to the wild type promoter, while others have a much less pronounced effect. A change of the A at position +4 into a G even results in a promoter up mutation. Remarkably, alteration of the A at position +1 into a G or a T affects the efficiency of initiation only slightly and initiation is maintained at the same position.

Characterization of the promoter of the large ribosomal RNA gene in yeast mitochondria and separation of mitochondrial RNA polymerase into two different functional components.

We have characterized a DNA sequence that functions in recognition of the promoter of the mitochondrial large rRNA gene by the yeast mtRNA polymerase. Promoter-containing DNA fragments were mutagenized and used as templates to study initiation of transcription in vitro with a partially purified mtRNA polymerase preparation. Deletion mutants, in which increasing stretches of DNA were removed from regions flanking the promoter, define a short area essential for correct initiation of transcription. It virtually coincides with a highly conserved stretch of nine nucleotides that is found immediately upstream of all transcriptional start sites described thus far. Two different point mutations within this nonanucleotide sequence drastically reduce promoter function. Conversely a single point mutation that results in the formation of a nonanucleotide sequence 99 nucleotides upstream of the large rRNA gene leads to a new, efficient transcription initiation site. MtRNA polymerase can be resolved into two different components by chromatography on Blue Sepharose: one retaining the capacity to synthesize RNA, the other conferring the correct specificity of initiation to the catalytic component.

In vitro site-directed mutagenesis with synthetic DNA oligonucleotides yields unexpected deletions and insertions at high frequency.

We have used in vitro site-directed mutagenesis with synthetic DNA oligonucleotides to introduce single nucleotide mutations in yeast mtDNA. In addition to the expected DNA alterations we also recovered with high frequency mutants with large deletions and insertions which arose through interaction with the synthetic DNA fragment. Characterization of a number of these by DNA sequence analysis has permitted reconstruction of the mutagenic events. In all cases, the DNA fragment had base paired with non-adjacent DNA sequences sometimes more than 1000 nucleotides apart from each other on the target strand. The products of such interactions cannot be avoided due to the non-stringent annealing conditions during complementary DNA strand synthesis. However, deliberate mispairing can be directed precisely, as shown by our ability to specifically delete the 1143-bp intron from the yeast mitochondrial gene coding for large ribosomal RNA with a synthetic DNA fragment consisting of the sequence of the exon borders flanking the intron.