Multiomics analysis of adaptation to repeated DNA damage in prostate cancer cells.

DNA damage is frequently utilized as the basis for cancer therapies; however, resistance to DNA damage remains one of the biggest challenges for successful treatment outcomes. Critically, the molecular drivers behind resistance are poorly understood. To address this question, we created an isogenic model of prostate cancer exhibiting more aggressive characteristics to better understand the molecular signatures associated with resistance and metastasis. 22Rv1 cells were repeatedly exposed to DNA damage daily for 6 weeks, similar to patient treatment regimes. Using Illumina Methylation EPIC arrays and RNA-seq, we compared DNA methylation and transcriptional profiles between the parental 22Rv1 cell line and the lineage exposed to prolonged DNA damage. Here we show that repeated DNA damage drives the molecular evolution of cancer cells to a more aggressive phenotype and identify molecular candidates behind this process. Total DNA methylation was increased while RNA-seq demonstrated these cells had dysregulated expression of genes involved in metabolism and the unfolded protein response (UPR) with Asparagine synthetase (ASNS) identified as central to this process. Despite the limited overlap between RNA-seq and DNA methylation, oxoglutarate dehydrogenase-like (OGDHL) was identified as altered in both data sets. Utilising a second approach we profiled the proteome in 22Rv1 cells following a single dose of radiotherapy. This analysis also highlighted the UPR in response to DNA damage. Together, these analyses identified dysregulation of metabolism and the UPR and identified ASNS and OGDHL as candidates for resistance to DNA damage. This work provides critical insight into molecular changes which underpin treatment resistance and metastasis.

Albumin phylogeny for clawed frogs (Xenopus).

Comparisons of albumin indicate that the frogs commonly used by North American molecular and developmental biologists under the name of Xenopus muelleri belong to another species, X. borealis. Phylogenetic analysis of the albumin data reveals two major groups of Xenopus species, one containing only X. tropicalis and the other, called the X. laevis grou, containing the remaining species of the genus. The phylogenetic tree, in conjunction with evidence from chromosomes and DNA content, leads to the hypothesis that total genome duplication occurred in the common ancestor of the X. laevis group.

Assessment of CXC ligand 12-mediated calcium signalling and its regulators in basal-like breast cancer cells.

CXC ligand (L)12 is a chemokine implicated in the migration, invasion and metastasis of cancer cells via interaction with its receptors CXC chemokine receptor (CXCR)4 and CXCR7. In the present study, CXCL12-mediated Ca2+ signalling was compared with two basal-like breast cancer cell lines, MDA-MB-231 and MDA-MB-468, which demonstrate distinct metastatic potential. CXCL12 treatment induced Ca2+ responses in the more metastatic MDA-MB-231 cells but not in the less metastatic MDA-MB-468 cells. Assessment of mRNA levels of CXCL12 receptors and their potential modulators in both cell lines revealed that CXCR4 and CXCR7 levels were increased in MDA-MB-231 cells compared with MDA-MB-468 cells. Cluster of differentiation (CD)24, the negative regulator of CXCL12 responses, demonstrated increased expression in MDA-MB-468 cells compared with MDA-MB-231 cells, and the two cell lines expressed comparable levels of hypoxia-inducible factor (HIF)2α, a CXCR4 regulator. Induction of epithelial-mesenchymal transition (EMT) by epidermal growth factor exhibited opposite effects on CXCR4 mRNA levels compared with hypoxia-induced EMT. Neither EMT inducer exhibited an effect on CXCR7 expression, however hypoxia increased HIF2α expression levels in MDA-MB-468 cells. Analysis of the gene expression profiles of breast tumours revealed that the highest expression levels of CXCR4 and CXCR7 were in the Claudin-Low molecular subtype, which is markedly associated with EMT features.

Oncosis and apoptosis induction by activation of an overexpressed ion channel in breast cancer cells.

The critical role of calcium signalling in processes related to cancer cell proliferation and invasion has seen a focus on pharmacological inhibition of overexpressed ion channels in specific cancer subtypes as a potential therapeutic approach. However, despite the critical role of calcium in cell death pathways, pharmacological activation of overexpressed ion channels has not been extensively evaluated in breast cancer. Here we define the overexpression of transient receptor potential vanilloid 4 (TRPV4) in a subgroup of breast cancers of the basal molecular subtype. We also report that pharmacological activation of TRPV4 with GSK1016790A reduced viability of two basal breast cancer cell lines with pronounced endogenous overexpression of TRPV4, MDA-MB-468 and HCC1569. Pharmacological activation of TRPV4 produced pronounced cell death through two mechanisms: apoptosis and oncosis in MDA-MB-468 cells. Apoptosis was associated with PARP-1 cleavage and oncosis was associated with a rapid decline in intracellular ATP levels, which was a consequence of, rather than the cause of, the intracellular ion increase. TRPV4 activation also resulted in reduced tumour growth in vivo. These studies define a novel therapeutic strategy for breast cancers that overexpress specific calcium permeable plasmalemmal ion channels with available selective pharmacological activators.

Partial characterization of different cell types found in the Xenopus laevis lymphoreticular tumor based on the presence or absence of surface immunoglobulins and Fc molecules.

Cells of Xenopus laevis lymphoreticular tumor induced by tumor tissue transplantation were examined for surface Ig and Fc receptor molecules in order to evaluate the different cell types found in the tumor. Direct immunofluorescent technique, using fluorochrome conjugated rabbit antisera to Xenopus Ig's, detected Ig molecules on the surface of a mean of 31.7 +/- 11.3% of cells in tumor suspensions. Most of these molecules were of IgM isotype, reversibly bound to the cell membrane (cytophilic) and could be dissociated by acid pH or overnight cell culturing. In addition integral membrane IgM was detected on the surface of 10.2 +/- 5.9% of the cells. The serum origin of cytophilic Ig's and the cellular origin of integral membrane Ig's were confirmed by analysis of electrophoretic mobility of their heavy chains on SDS-polyacrylamide gels. The existence of Fc receptor molecules on the surface of 48.6 +/- 16.6% of the cells was demonstrated by fluorescent staining using heat aggregated FITC labelled IgM or FITC or TRITC labelled antigen-complexed IgY antibodies. 32.2 +/- 12.4% and 16.4 +/- 6.8% of the cells bore receptors for IgY or receptors for IgM respectively, while 6.3 +/- 3.1% carried receptors for both Ig's. Double fluorescent staining revealed that 28.9 +/- 4.5% of cells bearing IgM on their surface expressed also receptors for IgY. These results attest to the heterogeneity of the tumor cell population, in respect to the presence or absence of FcR-IgY, FcR-IgM, sIgM, and cytophilic IgM surface molecules.

Biochemical studies of Xenopus laevis lymphocytes surface immunoglobulins.

Cell surface immunoglobulins of adult Xenopus laevis splenic small lymphocytes were analysed utilizing direct immunofluorescent staining and lactoperoxidase-catalysed radioiodination followed by immunoprecipitation of Ig molecules and characterization on SDS-PAGE. Nearly 30% of splenic lymphocytes are surface Ig positive. The HMW and LMW Ig classes are present on the surface of 23% and less than 5% of the cells, respectively. The mu chains of membrane HMW Ig have an apparent m.w. of 84,000 versus 73,000 for the mu chains of serum HMW Ig. Using immunofluorescent technique, we previously reported the absence of Ig molecules on the surface of larval Xenopus thymocytes. When the lactoperoxidase radioiodination technique was used, no cell surface Ig molecules could be isolated from Xenopus thymocytes.

B-lymphocyte populations in Xenopus laevis.

Two-color immunofluorescence technique was used to show the development and distribution of surface mu- cytoplasmic mu+ (s mu- c mu+) pre-B, s mu+ B- and s mu+ cIg+ plasma cells in metamorphic, postmetamorphic, and adult Xenopus. Generation of pre-B cells was evident in hematopoietic liver and spleen, but not in bone marrow, thymus, and duodenal mucosa. Surface immunoglobulin positive small lymphocytes were the most abundant in the spleen while plasma cells were detected in the thymus, duodenal mucosa, spleen, and liver. We had shown previously the appearance of s mu- c mu+ pre-B cells in the liver of Xenopus larvae at developmental stage 46 and later at stage 49 in the spleen. The frequency of pre-B cells dropped to zero at stage 58, the climax of metamorphosis. Pre-B cells start to reappear slowly as a second wave, at stage 60 through early postmetamorphic life in the liver and spleen. The percentage of surface Ig+ (sIg+) cells in the spleen of developing animals from stage 60 onward is comparable to that observed in adult life. In adult animals, the periphery of the liver continues to be active in hematopoiesis and contains some IgM producing plasma cells and rare sIg+ small lymphocytes while the pre-B cells are almost nonexistent in this region. The spleen, which is also active in some hematopoiesis, constitutes the main site of B-cell differentiation. Three ontogenic stages of pre-B, B-, and plasma cells are present in this organ. Pre-B and plasma cells are of low density and heterogeneous in size while small sIg+ B lymphocytes are of high density and much more homogeneous in size. The bone marrow in these lower anuran amphibia is rudimentary and is not a lymphopoietic tissue; in adult animals it is active only in differentiation of neutrophilic granulocytes.

Tryptic digestion of Xenopus IgM and IgY molecules.

Xenopus IgM and IgY molecules were digested by trypsin. Their respective fragments were separated by gel filtration and immunoadsorption. The purified fragments were characterized by SDS-PAGE and immunoblotting. Tryptic digestion of Xenopus IgM resulted in the release, at a low yield, of hexameric Fcmu, and of monovalent Fabmu fragments. The digestion of Xenopus IgY antibodies led to the recovery of divalent and monovalent Fab nu fragments. The antigen-binding property of these fragments was demonstrated. No Fc nu fragments of appreciable size could be detected.

Calcium influx pathways in breast cancer: opportunities for pharmacological intervention.

Ca(2+) influx through Ca(2+) permeable ion channels is a key trigger and regulator of a diverse set of cellular events, such as neurotransmitter release and muscle contraction. Ca(2+) influx is also a regulator of processes relevant to cancer, including cellular proliferation and migration. This review focuses on calcium influx in breast cancer cells as well as the potential for pharmacological modulators of specific Ca(2+) influx channels to represent future agents for breast cancer therapy. Altered expression of specific calcium permeable ion channels is present in some breast cancers. In some cases, such changes can be related to breast cancer subtype and even prognosis. In vitro and in vivo models have now helped identify specific Ca(2+) channels that play important roles in the proliferation and invasiveness of breast cancer cells. However, some aspects of our understanding of Ca(2+) influx in breast cancer still require further study. These include identifying the mechanisms responsible for altered expression and the most effective therapeutic strategy to target breast cancer cells through specific Ca(2+) channels. The role of Ca(2+) influx in processes beyond breast cancer cell proliferation and migration should become the focus of studies in the next decade.

Induction of epithelial-mesenchymal transition (EMT) in breast cancer cells is calcium signal dependent.

Signals from the tumor microenvironment trigger cancer cells to adopt an invasive phenotype through epithelial-mesenchymal transition (EMT). Relatively little is known regarding key signal transduction pathways that serve as cytosolic bridges between cell surface receptors and nuclear transcription factors to induce EMT. A better understanding of these early EMT events may identify potential targets for the control of metastasis. One rapid intracellular signaling pathway that has not yet been explored during EMT induction is calcium. Here we show that stimuli used to induce EMT produce a transient increase in cytosolic calcium levels in human breast cancer cells. Attenuation of the calcium signal by intracellular calcium chelation significantly reduced epidermal growth factor (EGF)- and hypoxia-induced EMT. Intracellular calcium chelation also inhibited EGF-induced activation of signal transducer and activator of transcription 3 (STAT3), while preserving other signal transduction pathways such as Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. To identify calcium-permeable channels that may regulate EMT induction in breast cancer cells, we performed a targeted siRNA-based screen. We found that transient receptor potential-melastatin-like 7 (TRPM7) channel expression regulated EGF-induced STAT3 phosphorylation and expression of the EMT marker vimentin. Although intracellular calcium chelation almost completely blocked the induction of many EMT markers, including vimentin, Twist and N-cadherin, the effect of TRPM7 silencing was specific for vimentin protein expression and STAT3 phosphorylation. These results indicate that TRPM7 is a partial regulator of EMT in breast cancer cells, and that other calcium-permeable ion channels are also involved in calcium-dependent EMT induction. In summary, this work establishes an important role for the intracellular calcium signal in the induction of EMT in human breast cancer cells. Manipulation of calcium-signaling pathways controlling EMT induction in cancer cells may therefore be an important therapeutic strategy for preventing metastases.

Distribution of immunoglobulin determinants on the surface of Xenopus laevis splenic lymphocytes.

The distribution of surface immunoglobulin (Ig) determinants on Xenopus laevis splenic lymphocytes after combination with divalent rabbit anti-Ig coupled to ferritin was studied. The electron micrographs showed the presence of immune complexes in 67% of lymphocytes treated at 0 degrees C-4 degrees C. The complexes were located all around the membrane and uniformly distributed in a random fashion. The variation of ferritin grain counts on cell sections is such, that the existence of two major subclasses of Ig-positive cells may be suggested. Raising the temperature produced a rapid interiorization of the complexes in vesicles without any previous aggregation to form a "cap" having occurred.

Structural studies of the Xenopus 19S immunoglobulin and 7S immunoglobulin and two immunoglobulin-like proteins.

Xenopus laevis 19S and 7S immunoglobulins (Ig) were extensively reduced and alkylated, their H and L chains spearated and their molecular weights determined. Two kinds of L chains of molecular weight 25,000 and 27,000 were revealed by SDS-polyacrylamide gel electrophoresis. In addition two Ig-like proteins consisting of heavy chains only, of 19S H-type and with similar molecular weight, were detected in Xenopus serum ans isolated. These proteins share common antigenic determinants with Xenopus 19S Ig heavy chains and are devoid of light chain determinants.