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The effect of droloxifene, a new anti-oestrogenic drug, on N-nitrosomethylurea-induced mammary tumours of Sprague-Dawley rats was investigated and compared with that of tamoxifen. The response of tumour growth to ovariectomy or to treatment with aminoglutethimide or high doses of oestradiol was also studied. Ovariectomy was by far the most effective treatment for mammary-tumour-bearing animals. More than 75% of the tumours in ovariectomized rats did not grow progressively but remained in remission for up to 12 weeks after castration when the experiment was terminated. The inhibitory effects of droloxifene and tamoxifen on mammary tumour growth were similar, but body weight loss of animals treated with tamoxifen was more marked than that of animals treated with droloxifene at the same dose and schedule. [ABSTRACT FROM AUTHOR]

In N-nitrosomethylurea-induced rat mammary tumours, tamoxifen is found to compete at the binding sites of the oestradiol receptor if a receptor determination is performed 1 day following the last drug application to animals. Despite a higher binding affinity of droloxifene (3-OH-tamoxifen) to oestradiol receptor, compared to tamoxifen, its influence on the measurable receptor quantity is only very weak or not demonstrable. Therefore, binding affinity is not a valid explanation for the different influences of the two anti-oestrogens on the receptor. These only can be attributed to different behaviour patterns of both substances in relation to their half-lives and metabolism and accumulation in the organism. Owing to the short half-life of droloxifene, even 1 day after the last application too little drug is available to compete for oestradiol binding sites. In the case of both anti-oestrogenic substances, cessation of drug application for 8 weeks abolished any influence on the oestradiol receptor. Furthermore, failure of aminoglutethimide to influence the oestradiol receptor could be observed because this substance does not act via this receptor. The experiments performed confirm literature data regarding the effect of aminoglutethimide therapy on oestradiol receptors breast tumour tissue of human beings. In summary: receptor investigations of N-nitrosomethylurea-induced rat mammary tumours, used as a model to test therapy regimens with droloxifene or other drugs with a short half-life, may be of limited value only. [ABSTRACT FROM AUTHOR]

The mainly Australian grass genus Austrostipa (tribe Stipeae) comprising approximately 64 species represents a remarkable example of an evolutionary radiation. To investigate aspects of diversification, macro- and micromorphological variation in this genus, we conducted molecular phylogenetic and scanning electron microscopy (SEM) analyses including representatives from most of Austrostipa 's currently accepted subgenera. Because of its taxonomic significance in Stipeae, we studied the lemma epidermal pattern (LEP) in 34 representatives of Austrostipa. Plastid DNA variation within Austrostipa was low and only few lineages were resolved. Nuclear ITS and Acc1 yielded comparable groupings of taxa and resolved subgenera Arbuscula , Petaurista , and Bambusina in a common clade and as monophyletic. In most of the Austrostipa species studied, the LEP was relatively uniform (typical maize-like), but six species had a modified cellular structure. The species representing subgenera Lobatae , Petaurista , Bambusina as well as A. muelleri from subg. Tuberculatae were well-separated from all the other species included in the analysis. We suggest recognizing nine subgenera in Austrostipa (with number of species): Arbuscula (4), Aulax (2), Austrostipa (36), Bambusina (2), Falcatae (10), Lobatae (5), Longiaristatae (2), Petaurista (2) and the new subgenus Paucispiculatae (1) encompassing A. muelleri . Two paralogous sequence copies of Acc1 , forming two distinct clades, were found in polyploid Austrostipa and Anemanthele . We found analogous patterns for our samples of Stipa s.str. with their Acc1 clades strongly separated from those of Austrostipa and Anemanthele . This underlines a previous hypothesis of Tzvelev (1977) that most extant Stipeae are of hybrid origin. We also prepared an up-to-date survey and reviewed the chromosome number variation for our molecularly studied taxa and the whole tribe Stipeae. The chromosome base number patterns as well as dysploidy and whole-genome duplication events were interpreted in a phylogenetic framework. The rather coherent picture of chromosome number variation underlines the enormous phylogenetic and evolutionary significance of this frequently ignored character., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Tkach, Nobis, Schneider, Becher, Winterfeld, Jacobs and Röser.)

[This corrects the article DOI: 10.1371/journal.pone.0192869.].

Karyotype characteristics can provide valuable information on genome evolution and speciation, in particular in taxa with varying basic chromosome numbers and ploidy levels. Due to its worldwide distribution, remarkable variability in morphological traits and the fact that ploidy change plays a key role in its evolution, the canary grass genus Phalaris (Poaceae) is an excellent study system to investigate the role of chromosomal changes in species diversification and expansion. Phalaris comprises diploid species with two basic chromosome numbers of x = 6 and 7 as well as polyploids based on x = 7. To identify distinct karyotype structures and to trace chromosome evolution within the genus, we apply fluorescence in situ hybridisation (FISH) of 5S and 45S rDNA probes in four diploid and four tetraploid Phalaris species of both basic numbers. The data agree with a dysploid reduction from x = 7 to x = 6 as the result of reciprocal translocations between three chromosomes of an ancestor with a diploid chromosome complement of 2n = 14. We recognize three different genomes in the genus: (1) the exclusively Mediterranean genome A based on x = 6, (2) the cosmopolitan genome B based on x = 7 and (3) a genome C based on x = 7 and with a distribution in the Mediterranean and the Middle East. Both auto- and allopolyploidy of genomes B and C are suggested for the formation of tetraploids. The chromosomal divergence observed in Phalaris can be explained by the occurrence of dysploidy, the emergence of three different genomes, and the chromosome rearrangements accompanied by karyotype change and polyploidization. Mapping the recognized karyotypes on the existing phylogenetic tree suggests that genomes A and C are restricted to sections Phalaris and Bulbophalaris, respectively, while genome B occurs across all taxa with x = 7.

Hybridization and polyploidization can radically impact genome organization from sequence level to chromosome structure. As a result, often in response to environmental change and species isolation, the development of novel traits can arise and will tend to result in the formation of homoploid or polyploid hybrid species. In this study we focus on evidence of hybridization and polyploidization by ascertaining the species parentage of the endemic alpine Helictotrichon parlatorei group. This group comprises five taxa; the diploids H. parlatorei, Helictotrichon setaceum subsp. setaceum and subsp. petzense, their putative hybrid Helictotrichon ×krischae and the hexaploid Helictotrichon sempervirens. For molecular analyses, cloned nuclear Topoisomerase VI genes of H. sempervirens and H. ×krischae were sequenced and compared with sequences of the diploids to estimate the evolutionary history in this group. In addition, detailed chromosome studies were carried out including fluorescence in situ hybridization (FISH) with 5S and 45S ribosomal and satellite DNA probes, and fluorochrome staining with chromomycin and DAPI. Two distinct types of Topoisomerase VI sequences were identified. One of them (SET) occurs in both subspecies of H. setaceum, the other (PAR) in H. parlatorei. Both types were found in H. ×krischae and H. sempervirens Karyotypes of H. parlatorei and H. setaceum could be distinguished by chromosomes with a clearly differentiated banding pattern of ribosomal DNAs. Both patterns occurred in the hybrid H. ×krischae Hexaploid H. sempervirens shares karyotype features with diploid H. parlatorei, but lacks the expected chromosome characteristics of H. setaceum, possibly an example of beginning diploidization after polyploidization. The geographic origin of the putative parental species and their hybrids and the possible biogeographical spread through the Alps are discussed., (Published by Oxford University Press on behalf of the Annals of Botany Company.)

Karyotype structures revealed by in situ hybridization with ribosomal and satellite DNAs and fluorochrome staining of AT- or GC-rich regions are reported for 23 diploid to tetraploid taxa of Aveneae genera Arrhenatherum, Avena, Helictotrichon, and Pseudarrhenatherum. Chromosomal features are compared with a molecular phylogeny generated on nuclear ribosomal (ITS, 5S) and chloroplast (matK) DNA sequences. Ancestral chromosomal character states are (1) two satellite chromosomes per set of x = 7, (2) 5S rDNA localized in nonsatellite chromosomes, (3) large chromosomes with (4) rather equal lengths of their respective chromosome arms, (5) sets with strong variance of chromosome lengths, (6) absence or small amounts of heterochromatin, and (7) absence or no detectable amplification of the satellite DNAs tested. Overall, most karyotype characteristics are species specific, but common patterns were found for the species of two large subgenera of Helictotrichon. Pseudarrhenatherum, although nested in the molecular phylogeny within Helictotrichon subgenus Helictotrichon, deviates strongly in karyotype characters such as Arrhenatherum as sister of Avena. The karyotype of Helictotrichon jahandiezii, sister to the clade of Helictotrichon subgenera Helictotrichon, Avena, and Arrhenatherum, strongly resembles that of Avena macrostachya. Karyotype features suggest that perennial A. macrostachya and H. jahandiezii are close to the C-genome species of annual Avena, whereas the Avena A genome resembles that of Arrhenatherum.

5S rDNA repeats studied in five genera of Aveneae have lengths between 285 and 329 bp (Avena sativa, Avena macrostachya, 26 species of Helictotrichon, Pseudarrhenatherum longifolium, Lagurus ovatus, and Trisetum spicatum). In only a single species (Helictotrichon aetolicum) an additional repeat of 456 bp occurs infrequently. Variation is largely due to insertions or deletions in the nontranscribed spacer as determined from sequences of 163 independent clones. The 5S gene of the Aveneae studied is conserved in length and sequence except for Helictotrichon bromoides and Helictotrichon marginatum in which duplications occur at two different sites. This new type of duplication and all duplications reported to date in 5S genes of angiosperms are shown to center on defined palindromic sequences. The "uncommon" 5S gene sequences detected in some Aveneae are not necessarily nonfunctional as pseudogenes because the essential features of the internal control region are maintained even after such duplication events. In each instance such gene sequences have spacers with unmodified structure, indicating that change in gene sequence is not necessarily coupled with change in adjacent spacers. The value of 5S spacer sequences for genomic identifications in Aveneae is exemplified in A. macrostachya (perennial), A. sativa (annual), and several diploid taxa of the genus Helictotrichon., (Copyright 2001 Academic Press.)

It was the aim of this study to compare drug-resistant sublines of the murine P388 in relation to resistance markers, the resistant phenotype and immunogenicity. Resistance to drugs either belonging to the MDR type (Doxorubicin, Vincristine, Mitoxantrone) or to the non-MDR type (Methotrexate) was generated in vivo in order to mimic the clinical situation. All resistant sublines expressed the mdr1 gene and the p-glycoprotein determined on m-RNA level or immunohistochemically, while no expression was registered in the parent P388. The rhodamine 123 fluorescence as marker for the energy dependent drug efflux pump was decreased only in the MDR-sublines, while the parent P388 and the Methotrexate-resistant line retained 100% or 90% of the dye, respectively. This indicates that the rhodamine efflux is a more function-related marker for MDR than the mdr1 gene and the pgp. The in vivo characterization of the sublines as regards their sensitivity to cytostatics revealed a clear-cut cross-resistance to MDR drugs in the MDR-lines, while the Methotrexate resistant subline was only cross-resistant to Cytarabine. In each resistant subline collateral sensitivity to certain but different cytostatics was observed. Experiments to overcome resistance by concomitant treatment with the modulators Nifedipine, Verapamil, Cyclosporin A and Chloroquin led to only limited success. The sublines P388/Mitox, P388/Vinc and P388/MTX developed immunogenicity which was never registered in the original P388. Vaccination with lethally irradiated drug-resistant cells resulted in a substantial rejection of viable tumor cells of the same line. With the P388/Mitox and P388/Vinc also an over-cross immunization was possible. This generation of immunogenicity as a concomitant characteristic of resistance should be considered as therapeutic potential also in the treatment of clinical cancer.

Continuing an earlier publication in which the growth of spontaneous mammary carcinomas in female mice of a C3H inbred strain was described, in the present paper the growth rate is expressed by a mathematical function, and is adapted by electronic data processing to the experimental values of 60 tumors classified into seven "growth types". For mathematical description the tumors were conveniently assumed to have a spherical shape, and the main reason for the reduction in volume increase with increasing tumor size was thought to be insufficient supply with oxygen and the essential nutrients S. Including the experimental data of all carcinomas investigated, the minimum concentration of S necessary for mitotic activity of the tumor cells was calculated to be 65% of the normal level. As the average for all the tumors investigated, a time of at least 33 hrs is required for the tumor volume to double.

In hematoxylin-eosin stained quatorial sections of 14 human bronchial tumors, the relative proportions of different tissue elements were determined in two perpendicular planes, 500 and 200 mu wide, using the target principle. Data characterizing the level of necrotization of these tumors have revealed a poor supply of oxygen and nutritive substances to tumors in which large stroma areas were alternating with large fields of tumor parenchyma. As anticipated, necrotization increases with increasing size of the tumor. A network of narrow stroma strips in the tumor affords a more favourable supply of the cells. The proportions of necrotic, hypoxic and GO areas were calculated.

Skin areas of the miniature pig were irradiated locally with single fractionated (10 X Df in 12 days) doses of 250 kVX-rays or 6.2 MeV neutron rays. Depending on the neutron dose for single irradiations, the acute skin reactions up to 4 months following exposure yielded RBE values significantly less than 3.0, related to a neutron dose of 8 Gy and an X-ray dose of 24.2 Gy. When giving fractionated doses, RBE values greater than 3.1 were obtained, related to a neutron dose fraction of 1.2 Gy and an X-ray dose fraction of 3.7 Gy. For late effects 2 years after a single exposure, RBE values significantly greater than 2.3 were obtained, related to a neutron dose of 8 Gy and an X-ray dose of 18.6 Gy. Fractionated neutron irradiation yielded, as with the acute damage, RBE values greater than 3.1, related to a neutron dose fraction of 1.2 Gy and an X-ray dose fraction of 3.7 Gy. The tendency to higher RBE values for late against acute skin reactions could not be statistically verified by the present results.

The incidence of spontaneous mammary carcinomas was determined in the C3H mouse inbred colony reared in Berlin-Buch over the past three years. Only female animals reaching a lifespan of no less than 170 days, corresponding to the longevity of the shortest lived animal in which a mammary carcinoma developed, were included in the study. A 50% incidence of mammary carcinomas was found with female animals above one year of age. The median age for the incidence of tumours was 446 days. Related to tumour-free females which at least have reached the median tumours age, the proportion of females developing mammary carcinomas was 86.8%. With increasing litters the tumour latency decreased from 491 for monoparous females to 383 days for mothers with 7 to 8 litters. The growth rate of 60 tumours under observation was found to vary grossly. With the minimum volume doubling time being the same in all cases, the growth rate of the tumours varied according to longer or shorter phases of stagnation or delay. Tumours having approximately equal growth rates throughout the period of investigation were classified as a "growth type". Altogether, seven growth types for spontaneous mammary carcinomas were established.