Intratumor heterogeneity of biomarker expression in breast carcinomas

Small biopsy samples are used increasingly to assess the biomarker expression for prognostic information and for monitoring therapeutic responses prior to and during neoadjuvant therapy. The issue of intratumor heterogeneity of expression of biomarkers, however, has raised questions about the validity of the assessment of biomarker expression based on limited tissue samples. We examined immunohistochemically the expression of HER-2neu (p185^erbB-2), epidermal growth factor receptor (EGFR), Bcl-2, p53, and proliferating cell nuclear antigen (PCNA) in 30 breast carcinomas using archived, paraffin embedded tissue and determined the extent of intratumor heterogeneity. Each section was divided into four randomly oriented discrete regions, each containing a portion of the infiltrating carcinoma. For each tumor, the entire lesion and four regions were analyzed for the expression of these markers. Scores of both membrane and cytoplasmic staining of HER-2neu and EGFR, scores of cytoplasmic staining of Bcl-2, and scores of nuclear staining of both p53 and PCNA were recorded. The intensity of staining and the proportion of immunostained cells were determined. A semiquantitative immunoscore was calculated by determining the sum of the products of the intensity and corresponding proportion of stained tumor cells. We analyzed both invasive (IDC) and in situ (DCIS) carcinomas. The Wilcoxon signed-rank test was used for paired comparisons between overall and regional immunoscores and between overall and regional percentages of stained cells. Spearman's correlation coefficients were used to assess the level of agreement of overall biomarker expression with each of the regions. Generalized linear models were used to assess overall and pair-wise differences in the absolute values of percent changes between overall and regional expression of biomarkers. For IDCs, there were no statistically significant differences in the expression of the biomarkers in terms of either the percentage of cells staining or the immunoscores when comparing the entire tumor with each region except for the lower EGFR expression of arbitrarily selected region 1 and lower p53 expression of region 1 compared to that of the entire tumor section. For DCIS, there were no statistically significant differences in the expression of the biomarkers between the entire tumor and each region except in PCNA of region 2 compared to that of entire tumor section. Positive correlation of immunoscores was observed between the entire tumor and each region as well as across all four regions for IDC. Similar observations were noted with DCIS except for HER-2neu and PCNA. No statistically significant differences were observed in the absolute values of percent changes of biomarker expression between overall and the four regions for both DCIS and IDC. Therefore, no significant intratumor heterogeneity in the expression of HER-2neu, Bcl-2, and PCNA was observed in IDC. Minor regional variations were observed for EGFR and p53 in IDC. Similarly, no significant regional variation in the expression of markers was observed in DCIS except for PCNA.     

Polychlorinated biphenyls disrupt cell division and tip growth in two species of fucoid algae

Environmental contaminants, including poly‐chlorinated biphenyls (PCBs), are enriched in coastal sediments, and despite a 1977 moratorium by the United States Environmental Protection Agency on the production of PCBs, levels remain high, more so near former industrial plants. The effects of these contaminants on sessile species in the intertidal zone, particularly nonanimal species such as the ubiquitous fucoid brown algae, are not well known. We investigated the developmental effects of chronic PCB treatment beginning at fertilization on two species of marine rockweed, Fucus vesiculosus Linnaeus and Silvetia compressa (J.Agardh) E.Serrão, T.O.Cho, S.M.Boo & Brawley. A mixture of the most widely used PCB congeners, Aroclors 1221, 1242, and 1254, was delivered at concentrations well below levels found in contaminated sediments, and resulted in severely delayed mitosis and cytokinesis in both species. In F. vesiculosus, this delay was accompanied by abnormal spindle morphology. PCB treatment also dramatically slowed or arrested rhizoid growth after 2–4 d, and by 7 d F. vesiculosus embryos were dead; in contrast, polar secretion of adhesive, germination, and photopolar germination were not affected. The dramatic delay in the first cell division and reduction in tip growth within the first week of development are likely to compromise S. compressa's ability to reproduce and establish new generations. Thus, the data presented here suggest that PCBs still present in coastal sediments may be inhibiting recruitment in these species. Moreover, as sediment dredging causes temporary spikes in PCB concentrations, these kinds of bioremediation steps may exacerbate the disruption of fucoid development.     

Quantitative analysis of DNA-damage response factors after sequential ion microirradiation

Several proteins are known to form foci at DNA sites damaged by ionizing radiation. We study DNA damage response by immunofluorescence microscopy after microirradiation of cells with energetic ions. By using microirradiation, it is possible to irradiate different regions on a single dish at different time-points and to differentiate between cells irradiated earlier and later. This allows to directly compare immunofluorescence intensities in both subsets of cells with little systematic error because both subsets are cultivated and stained under identical conditions. In addition, by using irradiation patterns such as crossing lines, it is possible to irradiate individual cells twice and to differentiate between immunofluorescence signals resulting from the cellular response to the earlier and to the later irradiation event. Here, we describe the quantitative evaluation of immunofluorescence intensities after sequential irradiation.     

Scanning irradiation device for mice in vivo with pulsed and continuous proton beams

A technical set-up for irradiation of subcutaneous tumours in mice with nanosecond-pulsed proton beams or continuous proton beams is described and was successfully used in a first experiment to explore future potential of laser-driven particle beams, which are pulsed due to the acceleration process, for radiation therapy. The chosen concept uses a microbeam approach. By focusing the beam to approximately 100 × 100 μm(2), the necessary fluence of 10(9) protons per cm(2) to deliver a dose of 20 Gy with one-nanosecond shot in the Bragg peak of 23 MeV protons is achieved. Electrical and mechanical beam scanning combines rapid dose delivery with large scan ranges. Aluminium sheets one millimetre in front of the target are used as beam energy degrader, necessary for adjusting the depth-dose profile. The required procedures for treatment planning and dose verification are presented. In a first experiment, 24 tumours in mice were successfully irradiated with 23 MeV protons and a single dose of 20 Gy in pulsed or continuous mode with dose differences between both modes of 10%. So far, no significant difference in tumour growth delay was observed.     

RAC1 regulates actin arrays during polarity establishment in the brown alga, Silvetia compressa

Multicellular development has evolved independently on numerous occasions and there is great interest in the developmental mechanisms utilized by each of the divergent lineages. Fucoid algae, in the stramenopile lineage (distinct from metazoans, fungi and green plants) have long been used as a model for early development based on unique life cycle characteristics. The initially symmetric fucoid zygote generates a developmental axis that determines not only the site of growth, but also the orientation of the first cell division, whose products have distinct developmental fates. Establishment and maintenance of this growth axis is dependent on formation of a filamentous actin array that directs vesicular movement, depositing new membrane and wall material for development of the rhizoid. What is not well known, is how formation and placement of the actin array is regulated in fucoid algae. A candidate for this function is Rac1, a small GTPase of the highly conserved Rho family, which has been implicated in controlling the formation of actin arrays in diverse eukaryotes. We demonstrate that Rac1 is not only present during formation of the filamentous actin array, but that its localization overlaps with the array in polarizing zygotes. Pharmacologically inhibiting Rac1 activity was shown to impede formation and maintenance of the actin array, and ultimately polar growth. Evidence is provided that a requirement of Rac1 function is its ability to associate with membranes via a post-translationally added lipid tail. Taken together, the data indicate that Rac1 is a necessary participant in establishment of the growth pole, presumably by regulating the placement and formation of the actin array. A role for Rac1 and related proteins in regulating actin is shared by animals, plants, fungi and with this work, brown algae, thus a conserved mechanism for generating polarity is in operation in unique eukaryotic lineages.     

Early evolution of metazoan serine/threonine and tyrosine kinases: identification of selected kinases in marine sponges

The phylum Porifera (sponges) was the first to diverge from the common ancestor of the Metazoa. In this study, six cDNAs coding for protein- serine/threonine kinases (PS/TKs) are presented; they have been isolated from libraries obtained from the demosponges Geodia cydonium and Suberites domuncula and from the calcareous sponge Sycon raphanus. Sequence alignments of the catalytic domains revealed that two major families of PS/TK, the "conventional" (Ca(2+)-dependent) protein kinase C (PKC), the cPKC subfamily, as well as the "novel" (Ca(2+)- independent) PKC (nPKC), form two separate clusters. In each cluster, the sequence from S. raphanus diverges first. To approach the question about the origin of protein-tyrosine kinases (PTK), which are found only in Metazoa, we analyzed two additional PS/TKs which have been cloned from S. domuncula: the stress-responsive protein kinase (KRSvSD) and the protein-kinase-C-related kinase (PRKvSD). The construction of the phylogenetic tree, comprising the eight PS/TKs and the PTK cloned previously from G. cydonium, revealed that the PTK derived from the branch including the KRSvSD kinase. These data facilitate the first molecular approach to elucidate the origin of metazoan PTK within the PS/TK superfamily.      

Regulation of rat ovarian cell growth and steroid secretion

A cultured rat ovarian cell line (31 A-F(2)) was used to study the effect of growth factors (epidermal growth factor [EGF] and fibroblast growth factor [FGF]), a survival factor (ovarian growth factor [OGF]), a hormone (insulin), and an iron-binding protein (transferring) on cell proliferation and steroid production under defined culture conditions. EGF and insulin were shown to be mitogenic (half-maximal response at 0.12 nM and 0.11 muM, respectively) for 31A-F(2) cells incubated in serum-free medium. EGF induced up to three doublings in the cell population, whereas insulin induced an average of one cell population doubling. FGF, OGF, and transferrin were found not to have any prominent effect on cell division when incubated individually with 31A-F(2) cells in serum-free medium. However, a combination of EGF, OGF, insulin, and transferrin stimulated cell division to the same approximate extent as cells incubated in the presence of 5 percent fetal calf serum. EGF or insulin did not significantly affect total cell cholesterol levels (relative to cells incubated in serum-free medium) when incubated individually with 31A-F(2) cells. However, cell cholesterol levels were increased by the addition of OGF (250 percent), FGF (370 percent), or a combination of insulin and EGF (320 percent). Progesterone secretion from 31A-F(2) cells was enhanced by EGF (25 percent), FGF (80 percent), and insulin (115 percent). However, the addition of a mitogenic mixture of EGF, OGF, insulin, and transferrin suppressed progesterone secretion 150 percent) below that of control cultures. These studies have permitted us to determine that EGF and insulin are mitogenic factors that are required for the growth of 31A-F(2) cells and that OGF and transferrin are positive cofactors that enhance growth. Also, additional data suggest that cholesterol and progesterone production in 31A-F(2) cells can be regulated by peptide growth factors and the hormone insulin.