Isoform specific changes in calcium transporters in colon and breast cancer cell lines

Calcium transporters play vital roles in the transport of calcium ions across cells of the mammary gland and the intestine. One such transporter is the plasma membrane Ca2+-ATPase (PMCA), of which there are 4 different genes (PMCA1-4). In these studies we investigated the hypothesis that the expression of PMCA is altered in HT-29 colon cancer cells during sodium butyrate and post-confluence mediated differentiation. We also investigated if PMCA expression is altered in breast cancer cell lines in an isofrom specific manner. Our results indicate isoform specific changes in PMCA mRNA and protein levels in HT-29 cells during differentiation, using real time RT-PCR and western blotting, respectively. We also observed pronounced alterations in the mRNA levels of the PMCA isoform linked to lactation (PMCA2) in a bank of breast cancer cell lines compared to normal cell lines. Changes in other isoforms were less pronounced. To further study the role of specific calcium transporters we have optimised conditions for the reverse transfection of MCF-7 breast cancer cells using NeoFX (Ambion). Using real time RT-PCR we have confirmed gene knockdown for specific isoforms and have studied the time course of knockdown over 96 hours. We see approximately 68 % inhibition at 24 hours increasing to 84 % 96 hours post-reverse transfection. Our studies suggest that the expression of specific calcium transporter isoforms can be significantly altered in cancer cell lines and that isoform specific inhibition of calcium transporters is possible using reverse transfection of siRNA

Molecular characterization of three gonad cell lines

To facilitate the study of the regulation and downstream interactions of genes involved in gonad development it is important to have a suitable cell culture model. We therefore aimed to characterize molecularly three different mouse gonad cell lines. TM3 and TM4 cells were originally isolated from prepubertal mouse gonads and were tentatively identified as being of Leydig cell and Sertoli cell origin, respectively, based upon their morphology and hormonal responses. The third line is a conditionally immortalized cell line, derived from 10.5-11.5 days post-coitum (dpc) male gonads of transgenic embryos carrying a temperature-sensitive SV40 large T-antigen. We studied by reverse transcription-polymerase chain reaction (RT-PCR) the expression profiles of a number of genes known to be important for early gonad development. Moreover, we assessed these cell lines for their capacity to induce Sox9 transcription upon expression of Sry, a key molecular event occurring during sex determination. We found that all three cell lines were unable to upregulate Sox9 expression upon transfection of Sry-expression constructs, even though these cells express many of the studied embryonic gonad genes. These observations point to a requirement for SRY cofactors for direct or indirect upregulation of Sox9 expression during testis determination. Copyright © 2003 S. Karger AG, Basel

The isoflavonoids genistein and quercetin activate different stress signaling pathways as shown by analysis of site-specific phosphorylation of ATM, p53 and histone H2AX

The ataxia-telangiectasia mutated (ATM) protein kinase is activated in response to ionizing radiation (IR) and activates downstream DNA-damage signaling pathways. Although the role of ATM in the cellular response to ionizing radiation has been well characterized, its role in response to other DNA-damaging agents is less well defined. We previously showed that genistein, a naturally occurring isoflavonoid, induced increased ATM protein kinase activity, ATM-dependent phosphorylation of p53 on serine 15 and activation of the DNA-binding properties of p53. Here. we show that genistein also induces phosphorylation of p53 at serines 6, 9, 20,46, and 392, and that genistein-induced accumulation and phosphorylation of p53 is reduced in two ATM-deficient human cell lines. Also, we show that genistein induces phosphorylation of ATM on serine 1981 and phosphorylation of histone H2AX on serine 139. The related bioflavonoids, daidzein and biochanin A, did not induce either phosphorylation of p53 or ATM at these sites. Like genistein, quercetin induced phosphorylation of ATM on serine 198 1, and ATM-dependent phosphorylation of histone H2AX on serine 139; however, p53 accumulation and phosphorylation on serines 6, 9, 15, 20, 46, and 392 occurred in ATM-deficient cells, indicating that ATM is not required for quercetin-induced phosphorylation of p53. Our data suggest that genistein and quercetin induce different DNA-damage induced signaling pathways that, in the case of genistein, are highly ATM-dependent but, in the case of quercetin, may be ATM-dependent only for some downstream targets. (C) 2003 Elsevier B.V. All rights reserved.

Tau haplotypes regulate transcription and are associated with Parkinson's disease

A primary haplotype (H1) of the microtubule-associated protein Tau (MAPT) gene is associated with Parkinson's disease (PD). However, the mechanism for disease susceptibility remains unknown. We examined the promoter region of MAPT and identified single nucleotide polymorphisms and insertions of 1 to 11 nucleotides. These polymorphisms corresponded to the previously characterized haplotypes, H1 and H2, as well as a novel variant of the H1 haplotype, H1'. As observed in other studies, we demonstrated a significant association with the H1/H1 promoter genotype and PD in a cohort of 206 idiopathic late-onset cases. This is in contrast with a panel of 13 early-onset PD patients, for whom we did not detect any mutations in MAPT. By examining single nucleotide polymorphisms in adjacent genes, we showed that linkage disequilibrium does not extend beyond the MAPT haplotype to neighboring genes. To define the mechanism of disease susceptibility, we examined the transcriptional activity of the promoter haplotypes using a luciferase reporter assay. We demonstrated in two human cell lines, SK-N-MC and 293, that the H1 haplotype was more efficient at driving gene expression than the H2 haplotype. Our data suggest that an increase in expression of the MAPT gene is a susceptibility factor in idiopathic PD.

Evolution of duplicate control regions in the mitochondrial genomes of metazoa: A case study with Australasian Ixodes ticks

To investigate the evolution pattern and phylogenetic utility of duplicate control regions (CRs) in mitochondrial (mt) genomes, we sequenced the entire mt genomes of three Ixodes species and part of the mt genomes of another I I species. All the species from the Australasian lineage have duplicate CRs, whereas the other species have one CR. Sequence analyses indicate that the two CRs of the Australasian Ixodes ticks have evolved in concert in each species. In addition to the Australasian Ixodes ticks, species from seven other lineages of metazoa also have mt genomes with duplicate CRs. Accumulated mtDNA sequence data from these metazoans and two recent experiments on replication of mt genomes in human cell lines with duplicate CRs allowed us to re-examine four intriguing questions about the presence of duplicate CRs in the mt genomes of metazoa: (1) Why do some mt genomes, but not others, have duplicate CRs? (2) How did mt genomes with duplicate CRs evolve? (3) How could the nucleotide sequences of duplicate CRs remain identical or very similar over evolutionary time? (4) Are duplicate CRs phylogenetic markers? It appears that mt genomes with duplicate CRs have a selective advantage in replication over mt genomes with one CR. Tandem duplication followed by deletion of genes is the most plausible mechanism for the generation of mt genomes with duplicate CRs. Once duplicate CRs occur in an mt genome, they tend to evolve in concert, probably by gene conversion. However, there are lineages where gene conversion may not always occur, and, thus, the two CRs may evolve independently in these lineages. Duplicate CRs have much potential as phylogenetic markers at low taxonomic levels, such as within genera, within families, or among families, but not at high taxonomic levels, such as among orders.

Quantitative analysis of MC1R gene expression in human skin cell cultures

To address the issue of melanocortin-1 receptor (MC1R) expression in non-melanocytic cells, we have quantitatively evaluated the relative expression levels of both MC1R mRNA and protein in a subset of different cell types. Using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) at high cycle numbers, we detected MC1R mRNA in all cell types examined, including human embryonic kidney-293 (HEK 293) cells, a cell type widely used as a negative control in melanocortin expression studies. Quantitative real-time PCR revealed the highest levels of MC1R transcripts were in melanocytic cells, whereas the keratinocyte and fibroblast cell cultures examined had only a low level of expression, similar to that of HEK 293 cells. Antibody mediated detection of MC1R protein in membrane extracts demonstrated exogenous receptor in MC1R transfected cell lines, as well as endogenous MC1R in melanoma cells. However, radioligand binding procedures were required to detect MC1R protein of normal human melanocytes and no surface expression of MC1R was detected in any of the non-melanocytic cells examined. This was consistent with their low level of mRNA, and suggests that, if present, the levels of surface receptor are significantly lower than that in melanocytes. The capacity of such limited levels of MC1R protein to influence non-melanocytic skin cell biology would likely be severely compromised. Indeed, the MC1R agonist [NIe(4), D-Phe(7)] alpha-melanocyte stimulating hormone (NDP-MSH) was unable to elevate intracellular cyclic adenosine monophosphate (cAMP) levels in the keratinocyte and fibroblast cells examined, whereas a robust increase was elicited in melanocytes. Although there are a variety of cell types with detectable MC1R mRNA, the expression of physiologically significant levels of the receptor may be more restricted than the current literature indicates, and within epidermal tissue may be limited to the melanocyte

The purine salvage enzyme hypoxanthine guanine xanthine phosphoribosyl transferase is a major target antigen for cell-mediated immunity to malaria

Although there is good evidence that immunity to the blood stages of malaria parasites can be mediated by different effector components of the adaptive immune system, target antigens for a principal component, effector CD4(+) T cells, have never been defined. We generated CD4+ T cell lines to fractions of native antigens from the blood stages of the rodent parasite, Plasmodium yoelii, and identified fraction-specific T cells that had a Th1 phenotype (producing IL-2, IFN-gamma, and tumor necrosis factor-a, but not IL-4, after antigenic stimulation). These T cells could inhibit parasite growth in recipient severe combined immunodeficient mice. N-terminal sequencing of the fraction showed identity with hypoxanthine guanine xanthine phosphoribosyl transferase (HGXPRT). Recombinant HGXPRT from the human malaria parasite, Plasmodium falciparum, activated the T cells in vitro, and immunization of normal mice with recombinant HGXPRT reduced parasite growth rates in all mice after challenge.

Characterization of the human sulfate anion transporter (hsat-1) protein and gene (SAT1; SLC26A1)

Sulfate plays an essential role during growth, development, bone/cartilage formation, and cellular metabolism. In this study, we have isolated the human sulfate anion transporter cDNA (hsat-1; SCL26A1) and gene (SAT1), determined its protein function in Xenopus oocytes and characterized SAT1 promoter activity in mammalian renal cell lines. hsat-1 encodes a protein of 75 kDa, with 12 putative transmembrane domains, that induces sulfate, chloride, and oxalate transport in Xenopus oocytes. hsat-1 mRNA is expressed most abundantly in the kidney and liver, with lower levels in the pancreas, testis, brain, small intestine, colon, and lung. The SAT1 gene is comprised of four exons stretching 6 kb in length, with an alternative splice site formed from an optional exon. SAT1 5' flanking region led to promoter activity in renal OK and LLC-PK1 cells. Using SAT1 5' flanking region truncations, the first 135 bp was shown to be sufficient for basal promoter activity. Mutation of the activator protein-1 (AP-1) site at position 252 in the SAT1 promoter led to loss of transcriptional activity, suggesting its requirement for SAT1 basal expression. This study represents the first functional characterization of the human SAT1 gene and protein encoded by the anion transporter hsat-1.

Antitumor activity of 3-ingenyl angelate: Plasma membrane and mitochondrial disruption and necrotic cell death

Options for skin cancer treatment currently include surgery, radiotherapy, topical chemotherapy, cryosurgery, curettage, and electrodes-sication. Although effective, surgery is costly and unsuitable for certain patients. Radiotherapy can leave a poor cosmetic effect, and current chemotherapy is limited by low cure rates and extended treatment schedules. Here, we describe the preclinical activity of a novel topical chemotherapeutic agent for the treatment of skin cancer, 3-ingenyl angelate (PEP005), a hydrophobic diterpene ester isolated from the plant Euphorbia peplus. Three daily topical applications of 42 nmol (18 mug) of PEP005 cured a series of s.c. mouse tumors (B16 melanoma, LK2 UV-induced squamous cell carcinoma, and Lewis lung carcinoma; it = >14 tumors/group) and human tumors (DO4 melanoma, HeLa cervical carcinoma, and PC3 and DU145 prostate carcinoma; it = >4 tumors/group) previously established (5-10 mm(3)) on C57BL/6 or Fox1(nu) mice. The treatment produced a mild, short-term erythema and eschar formation but, ultimately, resulted in excellent skin cosmesis. The LD90 for PEP005 for a panel of tumor cell lines was 180-220 muM. Electron microscopy showed that treatment with PEP005 both ill vitro (230 tot) and ill vivo (42 nmol) rapidly caused swelling of mitochondria and cell death by primary necrosis. Cr-51 release, uptake of propidium iodide, and staining with the mitochondria dye JC1, revealed that PEP005 (230 muM) treatment of tumor cells ill vitro resulted in a rapid plasma membrane perturbation and loss of mitochondrial membrane potential. PEP005 thus emerges as a new topical anti-skin cancer agent that has a novel mode of action involving plasma membrane and mitochondrial disruption and primary necrosis, ultimately resulting in an excellent cosmetic outcome.

Characterisation of human kallikrein 6 protease M expression in ovarian cancer

Kallikrein 6 (hK6, also known as protease M/zyme/neurosin) is a member of the human kallikrein gene family. We have previously cloned the cDNA for this gene by differential display and shown the overexpression of the mRNA in breast and ovarian primary tumour tissues and cell lines. To thoroughly characterise the expression of this kallikrein in ovarian cancer, we have developed a novel monoclonal antibody specific to hK6 and employed it in immunohistochemistry with a wide range of ovarian tumour samples. The expression was found elevated in 67 of 80 cases of ovarian tumour samples and there was a significant difference in the expression levels between normal and benign ovarian tissues and the borderline and invasive tumours (P<0.001). There was no difference of expression level between different subtypes of tumours. More significantly, high level of kallikrein 6 expression was found in many early-stage and low-grade tumours, and elevated hK6 proteins were found in benign epithelia coexisting with borderline and invasive tissues, suggesting that overexpression of hK6 is an early phenomenon in the development of ovarian cancer. Quantitative real-time reverse transcription-polymerase chain reactions also showed elevated kallikrein 6 mRNA expression in ovarian tumours. Genomic Southern analysis of 19 ovarian tumour samples suggested that gene amplification is one mechanism for the overexpression of hK6 in ovarian cancer.