• Mannosomes: a molluscan intracellular tubular membrane system related to heavy metal stress?

      Knigge, Thomas; Mann, Neelam; Parveen, Zahida; Perry, Christopher; Gernhöfer, Maike; Triebskorn, Rita; Köhler, Heinz-R; Connock, Martin (Elsevier BV, 2002)
      Amongst animals, several hydrogen peroxide-generating oxidases are apparently restricted to molluscs. One of these, -mannitol oxidase, is concentrated in the alimentary system, where it is associated with its own subcellular membrane system of unique tubular morphology, most likely representing a structural modification of the ER. These structures can be purified by subcellular fractionation and have been termed ‘mannosomes’. Little is known about the functions of mannitol oxidase or of mannosomes, but the previously reported molluscicide-induced increase in mannosomes implies their involvement in a general stress reaction. In this study, we examined the effects of heavy metal stress in the terrestrial gastropod Arion lusitanicus. The activity of mannitol oxidase and mannosome abundance were monitored, together with metal effects on heat-shock protein level, and these parameters were compared to heavy metal accumulation in the digestive gland. We found that mannitol oxidase is inhibited by heavy metals more than other oxidases. On the other hand, hsp70 levels and mannosomal protein were increased with enhanced heavy metal stress, the latter indicating a probable increase in the number of mannosome organelles. Thus, stress protein (hsp70) and mannosomal protein were positively correlated with heavy metal accumulation, whereas the enzyme activity showed a negative correlation with increasing heavy metal content of the slugs.
    • Mechanistic and predictive profiling of 5-Fluorouracil resistance in human cancer cells.

      Wang, Weiguang; Cassidy, James; O'Brien, Vincent; Ryan, Kevin; Collie-Duguid, Elaina (American Association for Cancer Research, 2004)
      Gene expression was analyzed in five pairs of 5-fluorouracil (5-FU) resistant and parental cancer cell lines on DNA microarrays. In unsupervised analysis, a prediction rule was built from the expression profiles of 29 genes, and 5-FU sensitivity class was predicted with 100% accuracy and high predictive strength. In supervised analysis of key 5-FU pathways, expression of 91 genes was associated with 5-FU sensitivity phenotype and segregated samples accordingly in hierarchical analysis. Key genes involved in 5-FU activation were significantly down-regulated (thymidine kinase, 2.9-fold; orotate phosphoribosyltransferase, 2.3-fold; uridine monophosphate kinase, 3.2-fold; pyrimidine nucleoside phosphorylase 3.6-fold) in resistant cells. Overexpression of thymidylate synthase and its adjacent gene, c-Yes, was detected in the resistant cell lines. The mRNA and protein overexpression of nuclear factor kappaB (NFkappaB) p65 and related antiapoptotic c-Flip gene was detected in resistant cells. The 5-FU-resistant cell lines also showed high NFkappaB DNA-binding activity. Cotransfection of NFkappaB p50 and p65 cDNA induced 5-FU resistance in MCF-7 cells. Both NFkappaB- and 5-FU-induced resistant cell lines manifested reduced expression of genes governing G(1)-S and S-phase transition. Expression of genes involved in DNA replication was also down-regulated in resistant cell lines. These findings were highly consistent with the slower growth rate, higher proportion of G(1), and lower proportion of S-phase cells in the resistant cell lines. This phenotype may protect resistant cells from cell death induced by incorporation of 5-FU into DNA chains, by allowing time to repair 5-FU-induced damage. Our findings may provide novel targets for tackling 5-FU resistance.
    • Messenger RNA expression profiling of genes involved in epidermal growth factor receptor signalling in human cancer cells treated with scanning array-designed antisense oligonucleotides.

      Petch, Amelia K.; Sohail, Muhammad; Hughes, Marcus D.; Benter, Ibrahim; Darling, John L.; Southern, Edwin M.; Akhtar, Saghir (Amsterdam: Elsevier, 2003)
      Scanning oligodeoxynucleotide (ODN) arrays appear promising in vitro tools for the prediction of effective antisense reagents but their usefulness has not yet been reported in mammalian systems. In this study, we have evaluated the use of scanning ODN arrays to predict efficacious antisense ODNs targeting the human epidermal growth factor receptor (EGFR) mRNA in a human epidermoid cancer cell line and in primary human glioma cells. Hybridisation accessibility profile of the first 120nt in the coding region of the human EGFR mRNA was determined by hybridising a radiolabelled EGFR transcript to a scanning array of 2684 antisense sequences ranging from monomers to 27-mers. Two ODNs, AS1 and AS2, complementary to accessible sequences within the EGFR mRNA, were designed and their ability to hybridise to EGFR mRNA was further confirmed by in vitro RNase H-mediated cleavage assays. Phosphorothioate-modified 21-mer AS1 and AS2 ODNs inhibited the growth of an established human A431 cancer cell line as well as primary glioma cells from human subjects when delivered as cationic lipoplexes. In contrast, scrambled controls and AS3-an antisense ODN complementary to an inaccessible site in EGFR mRNA-were inactive. Western blots showed that AS1 ODN exhibited a dose-dependent inhibition of EGFR protein expression in A431 cells in the nanomolar range. Microarray-based gene expression profiling studies of A431 cells treated with the 21-mer phosphorothioate AS1 ODN demonstrated successful inhibition of downstream signalling molecules further confirming the effective inhibition of EGFR expression in human cancer cells by antisense ODNs designed by scanning ODN array technology.
    • MGMT methylation status and expression level do not correlate with sensitivity to CCNU in short-term cultures derived from malignant astrocytoma

      Warr, Tracy; Poh, R.; Suarez-Merino, Blanca; Ward, Samantha; Warren, Phil; Darling, John L.; Thomas, David G. (Society for Neuro-Oncology and Duke University Press, 2005)
      Adjuvant chemotherapy using DNA-damaging agents has largely failed to make a significant impact on the outcome of patients with malignant astrocytoma. One of the primary mechanisms of resistance to nitrosureas such as CCNU is mediated through O6-methylguanine-DNA methyltrans-ferase (MGMT). This DNA repair enzyme removes the cytotoxic alkyl adducts from O6-guanine, and hence the level of MGMT activity in tumor cells is related to their sensitivity to nitroureas. It has been proposed that functional inactivation of MGMT through hypermethylation of the gene promotor region could be predictive of chemosensitivity. We have previously reported differential sensitivity to CCNU in a panel of 17 short-term cultures derived from malignant astrocytoma. In this study, we determined the methylation status of MGMT using methylation-specific PCR in these 17 cultures. We also assessed the amounts of MGMT mRNA and protein present in each culture using real-time quantitative PCR and immunohistochemistry with a commercial antibody against MGMT. There was good correlation between MGMT promotor methylation and presence of MGMT mRNA and protein in all but 2 cases. In both these cultures, mRNA and protein were not detected even though the MGMT promotor was unmethylated. However, there was no correlation between sensitivity to CCNU and MGMT status. In the 2 most resistant cultures, the MGMT gene was methylated and was not expressed. Similarly, in 4/5 of the most sensitive cultures, MGMT was unmethylated, and in 2 of these cases, there was commensurate MGMT expression. However, in the remaining 2 cultures, MGMT expression was not detected, indicating that an alternative mechanism to gene methylation is responsible for MGMT inactivation. This study highlights that the resistance of malignant astrocytoma to nitroureas may be more complex than simple reliance on MGMT activity and prediction of response to such agents by MGMT methylation status should be used with caution.
    • Mitoparan and target-selective chimeric analogues: membrane translocation and intracellular redistribution induces mitochondrial apoptosis.

      Jones, Sarah; Martel, Cecile; Belzacq-Casagrande, Anne-Sophie; Brenner, Catherine; Howl, John D. (Amsterdam: Elsevier, 2008)
      Mastoparan, and structurally-related amphipathic peptides, may induce cell death by augmentation of necrotic and/or apoptotic pathways. To more precisely delineate cytotoxic mechanisms, we determined that [Lys(5,8)Aib(10)]mastoparan (mitoparan) specifically induces apoptosis of U373MG and ECV304 cells, as demonstrated by endonuclease and caspase-3 activation and phosphatidylserine translocation. Live cell imaging confirmed that, following translocation of the plasma membrane, mitoparan specifically co-localizes with mitochondria. Complementary studies indicated that mitoparan induces swelling and permeabilization of isolated mitochondria, through cooperation with a protein of the permeability transition pore complex VDAC, leading to the release of the apoptogenic factor, cytochrome c. N-terminal acylation of mitoparan facilitated the synthesis of chimeric peptides that incorporated target-specific address motifs including an integrin-specific RGD sequence and a Fas ligand mimetic. Significantly, these sychnologically-organised peptides demonstrated further enhanced cytotoxic potencies. We conclude that the cell penetrant, mitochondriotoxic and apoptogenic properties of mitoparan, and its chimeric analogues, offer new insights to the study and therapeutic induction of apoptosis.
    • Mitoparans: mitochondriotoxic cell penetrating peptides and novel inducers of apoptosis.

      Jones, Sarah; Martel, Cecile; Belzacq-Casagrande, Anne-Sophie; Brenner, Catherine; Howl, John D. (Australian Peptide Association, 2007)
      Introduction: The amphipathic helical peptide mastoparan (MP; H-INLKALAALAKKIL-NH2) inserts into biological membranes to modulate the activity of heterotrimeric G proteins and other targets. Moreover, whilst cell free models of apoptosis demonstrate MP to facilitate mitochondrial permeability transition and release of apoptogenic cytochrome c, MP-induced death of intact cells has been attributed to its non-specific membrane destabilising properties (necrotic mechanisms). However, MP and related peptides are known to activate other signalling systems, including p42/p44 MAP kinases and could therefore, also modulate cell fate and specific apoptotic events. The ability of MP to facilitate mitochondrial permeability in cell free systems has lead to proposals that MP could be of utility in tumour therapeutics provided that it conferred features of cellular penetration and mitochondrial localization. We have recently reported that our highly potent amphipathic MP analogue mitoparan (mitP; [Lys5,8Aib10]MP; Aib = -aminoisobutyric acid) specifically promotes apoptosis of human cancer cells, as was confirmed by in situ TUNEL staining and activation of caspase-3. Moreover, we have also demonstrated that mitP penetrates plasma membranes and redistributes to co-localize with mitochondria. Complementary studies, using isolated mitochondria, further demonstrated that mitP, through co-operation with a protein of the permeability transition pore complex voltage-dependent anion channel (VDAC), induced swelling and permeabilization of mitochondria, leading to the release of the apoptogenic factor cytochrome c. An expanding field of peptide and cell penetrating peptide (CPP) research has focussed on the selective targeting of tumours by engineering constructs that incorporate cell-specific or tissue–specific address motifs. Peptidyl address motifs could enhance the selectivity of drug delivery whilst the improved cellular uptake offered by CPP enhances bioavailability. Thus and as a potential therapeutic strategy, we extended our findings to design target-specific mitP analogues. The integrin-specific address motif RGD and a Fas ligand mimetic WEWT were incorporated by N-terminal acylation of mitP to produce novel tandem-linked chimeric peptides.
    • Molecular pathology of brain tumours - how will molecular and cell biology contribute to improved outcomes in patients with malignant brain tumours?

      Darling, John L. (Czech Republic: Palacky University, Olomouc: Faculty of Medicine and Dentistry, 2006)
      Although in comparison to breast, lung and colon cancer, the brain is a relatively uncommon site for the development of cancer, the brain is the tenth most common site for the development of cancer in men and about the twelfth in women. This translates to about 6,000 individuals in the UK developing a primary malignant brain tumour every year. Cancer of the brain develops in two distinct age groups, although the types of tumour that develop in these two age groups differ markedly. There is a peak of incidence in the first decade of life, and brain tumours rank with leukaemia as a leading cause of cancer death in children. These tumours tend to be indolent low-grade astrocytomas or highly malignant primitive neuroectodermal tumours like medulloblastoma. However, the vast majority of brain tumours occur with increasing frequency in the sixth, seventh and eight decade of life and they are the second fastest growing cause of cancer death among those over 65. These tumours tend to be malignant astrocytomas particularly the most malignant variety, glioblastoma multiforme (GBM). Unlike lung cancer or malignant melanoma there is no strong evidence of an environmental carcinogen associated with the development of these tumours and no change in behaviour reduces risk.
    • Monoclonal antibodies: The tools of the trade within biomedical science

      Nelson, Paul N.; Astley, S.J.; Warren, Phil (John Wiley & Sons, Ltd., 2003)
      This book: The latest edition of this highly successful text, covers the major advances in the methods used in cellular and molecular pathology. In recent years, knowledge of the molecular organization of the cell has led to the development of powerful new techniques that bring greater accuracy and objectives to the diagnosis, prognosis and management of many diseases and to the study of pathological states. This book describes the latest molecular techniques available for the analysis of diseases. In particular it includes new techniques using fluorescent dyes, DNA microarrays, protein chemistry, and mass spectrometry. It also incorporates information from the Human Genome Project, and the new disciplines of genomics and proteomics, where relevant to pathology. Color plates are a new feature of this edition, illustrating the advances in fluorescence labeling of cells.
    • Neuronal Nitric Oxide Synthase Signaling in the Heart Is Regulated by the Sarcolemmal Calcium Pump 4b.

      Oceandy, Delvac; Cartwright, Elizabeth J.; Emerson, Michael; Prehar, Sukhpal; Baudoin, Florence M.; Zi, Min; Alatwi, Nasser; Venetucci, Luigi; Schuh, Kai; Williams, Judith C.; et al. (American Heart Association, 2007)
      BACKGROUND: Neuronal nitric oxide synthase (nNOS) has recently been shown to be a major regulator of cardiac contractility. In a cellular system, we have previously shown that nNOS is regulated by the isoform 4b of plasma membrane calcium/calmodulin-dependent ATPase (PMCA4b) through direct interaction mediated by a PDZ domain (PSD 95, Drosophilia Discs large protein and Zona occludens-1) on nNOS and a cognate ligand on PMCA4b. It remains unknown, however, whether this interaction has physiological relevance in the heart in vivo. METHODS AND RESULTS: We generated 2 strains of transgenic mice overexpressing either human PMCA4b or PMCA ct120 in the heart. PMCA ct120 is a highly active mutant form of the pump that does not interact with or modulate nNOS function. Calcium was extruded normally from PMCA4b-overexpressing cardiomyocytes, but in vivo, overexpression of PMCA4b reduced the beta-adrenergic contractile response. This attenuated response was not observed in ct120 transgenic mice. Treatment with a specific nNOS inhibitor (Nomega-propyl-L-arginine) reduced the beta-adrenergic response in wild-type and ct120 transgenic mice to levels comparable to those of PMCA4b transgenic animals. No differences in lusitropic response were observed in either transgenic strain compared with wild-type littermates. CONCLUSIONS: These data demonstrate the physiological relevance of the interaction between PMCA4b and nNOS and suggests its signaling role in the heart.
    • Non-Steroidal Anti-Inflammatory Drugs, DNA Repair and Cancer

      Dibra, Harpreet K.; Perry, Chris J.; Nicholl, Iain D. (InTech, 2011)
    • Novel functional interaction between the plasma membrane Ca2+ pump 4b and the proapoptotic tumor suppressor Ras-associated factor 1 (RASSF1).

      Armesilla, Angel Luis; Williams, Judith C.; Buch, Mamta H.; Pickard, Adam; Emerson, Michael; Cartwright, Elizabeth J.; Oceandy, Delvac; Vos, Michele D.; Gillies, Sheona; Clark, Geoffrey J.; et al. (American Society for Biochemistry and Molecular Biology, 2004)
      Plasma membrane calmodulin-dependent calcium ATPases (PMCAs) are enzymatic systems implicated in the extrusion of calcium from the cell. We and others have previously identified molecular interactions between the cytoplasmic COOH-terminal end of PMCA and PDZ domain-containing proteins. These interactions suggested a new role for PMCA as a modulator of signal transduction pathways. The existence of other intracellular regions in the PMCA molecule prompted us to investigate the possible participation of other domains in interactions with different partner proteins. A two-hybrid screen of a human fetal heart cDNA library, using the region 652-840 of human PMCA4b (located in the catalytic, second intracellular loop) as bait, revealed a novel interaction between PMCA4b and the tumor suppressor RASSF1, a Ras effector protein involved in H-Ras-mediated apoptosis. Immunofluorescence co-localization, immunoprecipitation, and glutathione S-transferase pull-down experiments performed in mammalian cells provided further confirmation of the physical interaction between the two proteins. The interaction domain has been narrowed down to region 74-123 of RASSF1C (144-193 in RASSF1A) and 652-748 of human PMCA4b. The functionality of this interaction was demonstrated by the inhibition of the epidermal growth factor-dependent activation of the Erk pathway when PMCA4b and RASSF1 were co-expressed. This inhibition was abolished by blocking PMCA/RASSSF1 association with an excess of a green fluorescent protein fusion protein containing the region 50-123 of RASSF1C. This work describes a novel protein-protein interaction involving a domain of PMCA other than the COOH terminus. It suggests a function for PMCA4b as an organizer of macromolecular protein complexes, where PMCA4b could recruit diverse proteins through interaction with different domains. Furthermore, the functional association with RASSF1 indicates a role for PMCA4b in the modulation of Ras-mediated signaling.
    • Novel mastoparan analogs induce differential secretion from mast cells.

      Farquhar, Michelle; Soomets, Ursel; Bates, Ruth L.; Martin, Ashley; Langel, Ulo; Howl, John D. (Elsevier Science B.V, 2002)
      Cationic amphiphilic peptides stimulate secretion via a receptor-independent action upon G proteins. We have previously utilized chimeric analogs of mastoparan (MP), including galparan (galanin(1-13)-MP ), as molecular probes of secretion. Here, we further resolve the structure-activity relationship of peptidyl secretagogs, including rationally designed chimeric MP analogs. The secretory efficacies of 10 MP analogs were significantly higher than 45 unrelated basic peptides. Comparative studies identified MP analogs that are differential secretagogs for 5-hydroxytryptamine (5-HT) and beta-hexosaminidase. Peptide-induced activation of phospholipase D (PLD), an enzyme intimately involved in regulated exocytosis [5], correlated with the secretion of beta-hexosaminidase but not 5-HT. Thus, these data indicate that different mechanisms are responsible for the exocytosis of 5-HT and beta-hexosaminidase, respectively. Moreover, mastoparan analogs are novel tools for probing the molecular details of exocytosis and other biological phenomena.
    • P-glycoprotein (MDR1) expression in leukemic cells is regulated at two distinct steps, mRNA stabilization and translational initiation.

      Yague, Ernesto; Armesilla, Angel Luis; Harrison, Georgina; Elliott, James; Sardini, Alessandro; Higgins, Christopher F.; Raguz, Selina (American Society for Biochemistry and Molecular Biology, 2003)
      Multidrug resistance in acute myeloid leukemia is often conferred by overexpression of P-glycoprotein, encoded by the MDR1 gene. We have characterized the key regulatory steps in the development of multidrug resistance in K562 myelogenous leukemic cells. Unexpectedly, up-regulation of MDR1 levels was not due to transcriptional activation but was achieved at two distinct post-transcriptional steps, mRNA turnover and translational regulation. The short-lived (half-life 1 h) MDR1 mRNA of naive cells (not exposed to drugs) was stabilized (half-life greater than 10 h) following short-term drug exposure. However, this stabilized mRNA was not associated with translating polyribosomes and did not direct P-glycoprotein synthesis. Selection for drug resistance, by long-term exposure to drug, led to resistant lines in which the translational block was overcome such that the stabilized mRNA was translated and P-glycoprotein expressed. The absence of a correlation between steady-state MDR1 mRNA and P-glycoprotein levels was not restricted to K562 cells but was found in other lymphoid cell lines. These findings have implications for the avoidance or reversal of multidrug resistance in the clinic.
    • Peptide Synthesis and Applications

      Howl, John D. (Clifton, N.J.: Humana Press, 2005)
      Hands-on experts describe in step-by-step detail the key methodologies of contemporary peptide synthesis and illustrate their numerous applications. The techniques presented include protocols for chemical ligation, the synthesis of cyclic and phosphotyrosine-containing peptides, lipoamino acid- and sugar-conjugated peptides, and peptide purification and analyses. Additional chapters detail methodologies and instrumentation for high-throughput peptide synthesis, many different applications of peptides as novel research tools and biological probes, and the design and application of fluorescent substrate-based peptides that can be used to determine the selectivity and activity of peptidases. A practical guide to the identification of proteins using mass spectrometric analyses of peptide mixtures is also included. (Humana Press)
    • Peptidyl-based delivery systems as a strategy for the therapeutic intervention of human astrocytoma and medulloblastoma

      Jones, Sarah; Howl, John D. (Springer Verlag, 2006)
      THIS BOOK: Understanding Biology Using Peptides: Proceedings of the 19th American Peptide Symposium highlights many of the recent developments in peptide science, with a particular emphasis on how these advances are being applied to basic problems in biology and medicine. Specific topics covered include novel synthetic strategies, peptides in biological signaling, post-translational modifications of peptides and proteins, peptide quaternary structure in material science and disease, and peptides as tools in drug discovery. (Springer Verlag)
    • Pharmacogenomic dissection of resistance to thymidylate synthase inhibitors.

      Wang, Weiguang; Marsh, S.; Cassidy, James; McLeod, Howard (American Association for Cancer Research, 2001)
      Chemoresistance is a major obstacle for successful cancer treatment. Gene amplification and altered expression are the main genetic mechanisms of tumor chemoresistance. Previously, only a limited number of genes were analyzed in each individual study using traditional molecular methods such as Northern and Southern blotting. In this study, the global gene expression patterns of 1176 genes in a panel of five thymidylate synthase (TS) inhibitor [raltitrexed (TDX) and 5-fluorouracil (5-FU)] resistant and sensitive parent cell lines were investigated using cDNA array technology. Only 28 of 1176 genes were altered >1.5-fold among resistant cells, with 2 genes (TS and YES1) consistently higher in the panel. TS mRNA and protein were consistently overexpressed in all drug-resistant tumor cell lines compared with the sensitive parent cell lines. Southern blot and FISH analysis demonstrated that the TS gene was amplified in 5-FU- and TDX-resistant cell lines. YES1 mRNA and protein were overexpressed in four drug-resistant tumor cell lines but were not overexpressed in the lymphoblast cell line W1L2(TDX), although the YES1 gene was highly amplified in these cells. The fact that W1L2 has high level (>10-fold) resistance to TS inhibitor in the absence of high YES1 expression leads to a conclusion that YES1 has no direct role in this drug resistance process. By narrowing the search from 1176 to 2 genes, the analysis of in vitro TDX and 5-FU resistance becomes more straightforward for confirmatory studies. These data provide encouragement that comprehensive transcript analysis will aid the quest for more enlightened therapeutics.
    • Phytoestrogens: perpetrators or protectors?

      Martin, Jan H.; Crotty, Stephen; Nelson, Paul N. (Future Medicine Ltd, 2007)
      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.
    • Picornavirus uncoating.

      Smyth, M.S.; Martin, Jan H. (British Medical Association, 2002)
      Recently, much has been learned about the molecular mechanisms involved in the pathogenesis of picornaviruses. This has been accelerated by the solving of the crystal structures of many members of this virus family. However, one stage of the virus life cycle remains poorly understood: uncoating. How do these simple but efficient pathogens protect their RNA genomes with a stable protein shell and yet manage to uncoat this genome at precisely the right time during infection? The purpose of this article is to review the current state of knowledge and the most recent theories that attempt to answer this question. The review is based extensively on structural data but also makes reference to the wealth of biochemical information on the topic.
    • Plasma membrane Ca2+ ATPase 4 is required for sperm motility and male fertility.

      Schuh, Kai; Cartwright, Elizabeth J.; Jankevics, Eriks; Bundschu, Karin; Liebermann, Jürgen; Williams, Judith C.; Armesilla, Angel Luis; Emerson, Michael; Oceandy, Delvac; Knobeloch, Klaus-Peter; et al. (American Society for Biochemistry and Molecular Biology, 2004)
      Calcium and Ca(2+)-dependent signals play a crucial role in sperm motility and mammalian fertilization, but the molecules and mechanisms underlying these Ca(2+)-dependent pathways are incompletely understood. Here we show that homozygous male mice with a targeted gene deletion of isoform 4 of the plasma membrane calcium/calmodulin-dependent calcium ATPase (PMCA), which is highly enriched in the sperm tail, are infertile due to severely impaired sperm motility. Furthermore, the PMCA inhibitor 5-(and-6)-carboxyeosin diacetate succinimidyl ester reduced sperm motility in wild-type animals, thus mimicking the effects of PMCA4 deficiency on sperm motility and supporting the hypothesis of a pivotal role of the PMCA4 on the regulation of sperm function and intracellular Ca(2+) levels.
    • Preoperative mitomycin, ifosfamide, and cisplatin followed by esophagectomy in squamous cell carcinoma of the esophagus: pathologic complete response induced by chemotherapy leads to long-term survival.

      Darnton, S.J.; Archer, V.R.; Stocken, D.D.; Mulholland, P.J.; Casson, A.G.; Ferry, David R. (American Society of Clinical Oncology, 2003)
      PURPOSE: Squamous cell carcinoma of the esophagus remains an aggressive disease with a poor prognosis, even after curative-intent surgery. This article analyzes the impact of preoperative chemotherapy with mitomycin, ifosfamide, and cisplatin (MIC) on a cohort of 68 patients. PATIENTS AND METHODS: From 1988 to 1994, 68 patients with potentially operable squamous cell carcinoma of the esophagus were entered onto two phase II trials of neoadjuvant chemotherapy with mitomycin 6 mg/m2, ifosfamide 3 g/m2, and cisplatin 50 mg/m2 and received between two and four cycles of treatment at 3-weekly intervals. Two patients were removed from the analysis when they were found to have malignancy other than squamous cell carcinoma of the esophagus. RESULTS: Forty (61%) of 66 patients had a radiologic response to chemotherapy (18 complete responses and 22 partial responses), and 52 (79%) of 66 patients went on to have the primary tumor resected. There were nine pathologic complete responders, seven of whom remain fit and well after at least 60 months of follow-up. The overall median survival was 12.4 months (95% confidence interval, 9.6 to 18.8 months). The complete response and node-negative patients survived significantly longer than those in other categories (log-rank chi2 = 18.8; P <.001): on average 13 months longer than the node-positive or nonresected category (22.0 v 9.4 months). The toxicity of the regimen was low. CONCLUSION: MIC is an easily administered, well-tolerated, and efficacious regimen as neoadjuvant therapy for patients with squamous cell carcinoma of the esophagus. These results warrant further investigation.