Jan Martin

Demystified . . . Human endogenous retroviruses

Human endogenous retroviruses (HERVs) are a family of viruses within our genome with similarities to present day exogenous retroviruses. HERVs have been inherited by successive generations and it is possible that some have conferred biological benefits. However, several HERVs have been implicated in certain cancers and autoimmune diseases. This article demystifies these retroviruses by providing an insight into HERVs, their means of classification, and a synopsis of HERVs implicated in cancer and autoimmunity. Furthermore, the biological roles of HERVs are explored.

Endothelial Nitric Oxide Synthase: Correlation with Histologic Grade, Lymph Node Status and Estrogen Receptor Expression in Human Breast Cancer

Nitric oxide is produced by several isoenzymes which are present in many different tissues. We have recently reported the presence of inducible nitric oxide synthase in a breast cancer cell line. The purpose of this study was to examine the distribution of endothelial nitric oxide synthase (eNOS) in a series of human breast tumours. Immunohistochemical investigations demonstrated immunolabelling of tumour cells with the primary antibody, bovine endothelial anti-nitric oxide synthase. Although there was no correlation between eNOS staining and tumour size, there was a significant (p < 0.005) negative correlation (ρ = –0.65) between the percentage of tumour cells staining positive for eNOS and the histologic grade of the tumour; there was also a significant (p < 0.05) negative correlation (ρ = –0.40) between the percentage of tumour cells staining positive for eNOS and the number of positive lymph nodes. A significant (p < 0.005) positive correlation (ρ = 0.63) between the percentage of tumour cells staining positive for eNOS and estrogen receptor (ER) expression by the tumour was also observed. In conclusion, our results demonstrate that eNOS is expressed by human breast tumours and that its presence negatively correlates with histologic grade and lymph node status and positively correlates with ER expression.

Human endogenous retroviruses: transposable elements with potential?

Human endogenous retroviruses (HERVs) are a significant component of a wider family of retroelements that constitute part of the human genome. These viruses, perhaps representative of previous exogenous retroviral infection, have been integrated and passed through successive generations within the germ line. The retention of HERVs and isolated elements, such as long‐terminal repeats, could have the potential to harm. In this review we describe HERVs within the context of the family of known transposable elements and survey these viruses in terms of superantigens and molecular mimics. It is entirely possible that these mechanisms provide the potential for undesired immune responses.

Divergent effect of taxol on proliferation, apoptosis and nitric oxide production in MHH225 CD34 positive and U937 CD34 negative human leukaemia cells

Paclitaxel (Taxol) has been shown to be clinically effective in treatment of patients with breast and ovarian cancer. It has also shown promising results in various other solid tumours. Paclitaxel has induced apoptosis in the G2/M phase of the cell cycle in both HL-60 and U937 human leukaemia cells. A recent study has shown a dose-dependent cytotoxicity for both taxanes: paclitaxel (taxol) and docetaxel (Taxotere) on fresh leukaemia cells in primary culture from 16 ALL and four AML patients and proposed their use in treatment of acute leukaemia patients. AML is a heterogeneous disease in which malignant transformation and disease progression occur at the level of CD34 positive cells. Also, the multi-drug resistance gene product, P-glycoprotein is expressed only in CD34 positive AML cells. Therefore, an in vitro evaluation of the efficacy of paclitaxel, a P-glycoprotein substrate, in CD34 positive AML cells is warranted before considering its clinical use in acute leukaemia patients. Since all in vitro studies of paclitaxel reported so far have involved only CD34 negative (HL-60, U937, K562) human AML cells, the aim of the present study was to evaluate paclitaxel efficacy against CD34 positive AML cells. The IC50 of paclitaxel for apoptosis was significantly higher in MHH225 CD34 positive cells (12 +/- 2 microM) than in U937 CD34 negative cells (1.7 +/- 0.2 microM), P < 0.001. Paclitaxel has a significantly weaker cytotoxic effect on CD34 positive AML cells. One log higher concentration of paclitaxel was required in MHH225 CD34 positive AML cells to achieve the same apoptosis level achieved in U937 CD34 negative leukaemia cells. Also, at the high concentration achievable in vivo: 10 microM paclitaxel, only half the MHH225 CD34 positive AML cells were apoptotic versus 72% of U937 CD34 negative leukaemia cells. Clearly, paclitaxel has only weak or modest in vitro efficacy compared with several conventional anti-leukaemia drugs used in AML treatment. The present results support the poor level of in vivo induction of apoptosis achieved during a phase I clinical study with paclitaxel therapy in 26 leukaemia patients. Also, the present results have shown a significant increase in nitric oxide production during paclitaxel-induced apoptosis in U937 monocytic leukaemia cells, confirming the vital role of nitric oxide in mediating paclitaxel-induced apoptosis by monocytic cells. In conclusion, the present study has demonstrated a clear difference between the effect of paclitaxel on CD34 negative and CD34 positive AML cells. Given its poor performance in the phase I clinical study of 26 acute leukaemia patients and the present weak in vitro cytotoxic effect, it is unlikely that paclitaxel will have a role in the treatment of acute leukaemia. Also, the present study emphasises the need to use CD34 positive AML cells such as MHH225 rather than the unsuitable lineage-specific CD34 negative cells such as HL-60 or U937 for in vitro pre-clinical screening of potential novel effective anti-leukaemia agents.

Identification of the pocket factors in a picornavirus

Summary. Bovine enterovirus (BEV), along with other enteroviruses and the rhinoviruses, has a hydrophobic pocket within structural protein VP1. In the crystal structures of these viruses there is electron density commensurate with a non-protein molecule within the pocket. These molecules, termed pocket factors, have been shown to stabilise the capsid and their removal from the pocket is a necessary prerequisite to uncoating. The pocket factors have been proposed, from the electron densities and uncoating studies, to be short chain fatty acids. In order to identify the pocket factor of BEV, we have grown and purified the virus in an identical manner to that used for the crystal structure determination and have performed a lipophilic extraction. Palmitic acid, C16:0, was the most abundant accounting for 40.8% by mass of the lipophilic extract (39.3 mol%). Myristic acid C14:0, was next most abundant at 18.5% by mass (20.0 mol%). In addition, we have identified other fatty acids in smaller proportions. We have therefore shown that BEV contains saturated fatty acid pocket factors of varying chain length. We have also compared the profile of the fatty acyl chain composition of BEV with those for uninfected BHK-21 cell plasma membrane and endoplasmic reticulum extracts.

Reduced expression of endothelial and inducible nitric oxide synthase in a human breast cancer cell line which has acquired estrogen independence.

We have recently reported the presence of inducible nitric oxide synthase (iNOS) in the human breast cancer cell line ZR-75-1. The purpose of the present study was to examine differences in expression of endothelial (eNOS) and inducible nitric oxide synthase in normal human mammary epithelial cells (HMEC) compared with two variants of the ZR-75-1 cell line. One variant has acquired estrogen independence, the other has acquired resistance to tamoxifen. Immunohistochemical investigations demonstrated that 100% of HMEC cells staining positive for both eNOS and iNOS. ZR-75-1 cells showed 100% staining for eNOS and 52% positive staining for iNOS. There was no difference in staining between the parent cell line and cells which had acquired resistance to tamoxifen (ZR-75-9a1). However, in the breast cancer cell line which had acquired estrogen independence (ZR-PR-LT), less than 5% of cells exhibited positive staining for eNOS and staining for iNOS was undetectable. L-Arginine increased NO production in both ZR-75-9a1 and ZR-PR-LT cells. Progesterone was able to down regulate NO production in both ZR-75-1 and ZR-75-9a1 cells and this effect was reversible by RU486. These results support the suggestion that loss of NOS expression may be associated with the progression of breast cancers.

A novel multiplex RT-PCR system detects human endogenous retrovirus-K in breast cancer

Summary.Human endogenous retrovirus HERV-K like-sequences have been implicated in certain cancers. We developed a novel multiplex RT-PCR system for HERV-K that yielded a 533 bp product together with a smaller sized product (319 bp) of the house keeping gene, histidyl tRNA synthetase (HtRNAS). The latter spanned an intron that also served to validate target cDNA. PCR amplicons of HERV-K and HtRNAS were visualised using a gel documentation system and the pixel intensity used to derive semi-quantitative levels of viral expression. Our data showed that HERV-K10 was significantly elevated in MCF-7 cells treated with estrogen. Interestingly, HERV-K expression was higher in MCF-7 cells selected with adriamycin. RT-PCR combined with Southern blotting also detected HERV-K from breast cancer tissue using laser capture microscopy. This study highlights the presence of HERV-K in the breast cancer cell lines MCF-7 and MCF-7 ADR and confirms HERV-K10 transcripts in the cell line T47D. We believe this study to be a novel approach in determining levels of HERV-K expression and for detecting this virus in cancer cell lines and tissues.

Phytoestrogens: perpetrators or protectors?

Phytoestrogens are estrogen-like substances produced by plants that account for some of the constituents present in vegetation that may be responsible for the health benefits of a diet rich in fruit and vegetables. Phytoestrogens have a plethora of different actions that they are capable of exerting on cellular metabolism. This review will focus on some of the major non-estrogen receptor-mediated cellular effects used by phytoestrogens and will draw attention to the fact that while they may have a number of beneficial effects, particularly in offering a protective effect against some hormone-dependent cancers, such as breast and prostate cancer, they may also have possible unfavorable effects by interfering with the functioning of normal cellular activities such as receptor-mediated signal transduction and DNA replication, as well as being genotoxic, mutagenic and promoting the proliferation of some cancer cells.

ZR-75-1 human breast cancer cells: expression of inducible nitric oxide synthase and effect of tamoxifen and phorbol ester on nitric oxide production

The existence of the L-arginine-nitric oxide pathway was investigated in ZR-75-1 human breast cancer cells. The presence of inducible nitric oxide synthase in these cells was confirmed by staining with an anti-iNOS antibody. ZR-75-1 cells spontaneously produced nitric oxide (NO) and this production could be significantly (P < 0.001) enhanced by L-arginine (0.01-10 mM) and was inhibited by L-NAME (2 mM). Stimulating the cells with phorbol 12-myristate 13-acetate (PMA) (200-1000 nM) resulted in a significant (P < 0.001) increase in NO2- secreted into the medium. Although treatment of the same cells with tamoxifen (10(-10)-10(-6) M) had no effect on NO production, tamoxifen was able to significantly (P < 0.001) downregulate PMA-enhanced nitrite production. Our results suggest that tamoxifen could play a role in the biology of nitric oxide in breast tumours.

High progesterone receptor concentration in a variant of the ZR-75-1 human breast cancer cell line adapted to growth in oestrogen free conditions.

Culture of ZR-75-1 human breast cancer cells for 5 days in the absence of oestrogens (phenol red-free medium supplemented with dextran coated charcoal stripped 5% fetal calf serum) resulted in a slowing of growth rate and loss of progesterone receptors. Oestradiol at 10(-9) M markedly stimulated growth and progesterone receptor synthesis over a 5-day period. While medroxyprogesterone acetate (10(-10) to 10(-6) M) inhibited growth of ZR-75-1 cells growing in complete medium, in the short-term absence of oestrogens low concentrations were growth stimulatory. Cells deprived of oestrogens for 5 days retained sensitivity to growth inhibition by 4-hydroxy tamoxifen. ZR-75-1 cells were also adapted to growth in the absence of oestrogens over a 5-month period. These cells (ZR-PR-LT) failed to express binding sites characteristic of the type 1 oestrogen receptor but progesterone receptor expression was at a level normally associated with oestrogen induction. Adapted cells were growth inhibited by oestradiol, 4-hydroxy tamoxifen and medroxyprogesterone acetate, but despite elevated progesterone receptor expression the progestin was only marginally more inhibitory than in the parent line. Our data indicate a poor quantitative relationship between response to progestins in vitro and progesterone receptor concentration and support previous findings that acquisition of an oestrogen independent phenotype does not necessarily result in resistance to anti-oestrogens.