Background:Immunodeficiency in ataxia telangiectasia (A-T) is less severe in patients expressing some mutant or normal ATM kinase activity. We, therefore, determined whether expression of residual ATM kinase activity also protected against tumour development in A-T.Methods:From a total of 296 consecutive genetically confirmed A-T patients from the British Isles and the Netherlands, we identified 66 patients who developed a malignant tumour; 47 lymphoid tumours and 19 non-lymphoid tumours were diagnosed. We determined their ATM mutations, and whether cells from these patients expressed any ATM with residual ATM kinase activity.Results:In childhood, total absence of ATM kinase activity was associated, almost exclusively, with development of lymphoid tumours. There was an overwhelming preponderance of tumours in patients <16 years without kinase activity compared with those with some residual activity, consistent with a substantial protective effect of residual ATM kinase activity against tumour development in childhood. In addition, the presence of eight breast cancers in A-T patients, a 30-fold increased risk, establishes breast cancer as part of the A-T phenotype.Conclusion:Overall, a spectrum of tumour types is associated with A-T, consistent with involvement of ATM in different mechanisms of tumour formation. Tumour type was influenced by ATM allelic heterogeneity, residual ATM kinase activity and age.

Three isoforms of the sarcoplasmic/endoplasmic reticulum Ca 2؉ ATPase (SERCA) are known to exist in mammalian cells. This study investigated the effects of thapsigargin and a variety of commonly used hydrophobic inhibitors on these SERCA isoforms (i.e. SERCA1b, SERCA2b, and SERCA3a), which were transiently expressed in COS-7 cells. In addition, the study assessed whether the introduction of the phenylalanine to valine mutation at position 256 (F256V), known to reduce the potency of thapsigargin inhibition in avian SERCA1, affects the other SERCA isoforms in a similar manner and whether this mutation also affects the inhibition by other inhibitors. This study has shown that the sensitivity to thapsigargin is different for the SERCA isoforms (apparent K i values being 0.21, 1.3, and 12 nM for SERCA1b, SERCA2b, and SERCA3a, respectively). The reduction in thapsigargin sensitivity caused by the F256V mutation was also different for the three isoforms, with SERCA2b only being modestly affected by this mutation. Although some of the other inhibitors investigated (i.e. cyclopiazonic acid and curcumin) showed some differences in their sensitivity toward the SERCA isoforms, most were little affected by the F256V mutation, indicating that they inhibit the Ca 2؉ -ATPase by binding to sites on SERCA distinct from that of thapsigargin.

No abstract

The sarcoplasmic-endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitors thapsigargin (0.1-1 μM) and cyclopiazonic acid (10 μM), failed to affect resting [Ca2+] in human spermatozoa. Slow progesterone-induced [Ca2+ i]i oscillations in human spermatozoa, which involve cyclic emptying-refilling of an intracellular Ca2+ store were also insensitive to these inhibitors. Non-selective doses of thapsigargin (5-30 μM, 50-300 times the saturating dose for SERCA inhibition), caused elevation of resting [Ca2+]i and partial, dose-dependent disruption of oscillations. A 10-40 μM concentration of bis(2-hydroxy-3-tert-butyl-5-methyl-phenyl)methane (bis-phenol), which inhibits both thapsigargin-sensitive and -insensitive microsomal Ca2+ ATPases, caused elevation of resting [Ca2+]i and inhibition of [Ca2+]i oscillations at doses consistent with inhibition of thapsigargin-resistant, microsomal ATPase and liberation of stored Ca2+. Low doses of bis-phenol had marked effects on [Ca2+]i oscillation kinetics. Application of the drug to cells previously stimulated with progesterone had effects very similar to those observed when it was applied to unstimulated cells, suggesting that the sustained Ca2+ influx induced by progesterone is not mediated via mobilisation of Ca2+ stores. Western blotting for human sperm proteins showed expression of secretory pathway Ca2+ ATPase (SPCA1). Immunolocalisation studies revealed expression of SPCA1 in all cells in an area behind the nucleus, extending into the midpiece. Staining for SERCA, carried out in parallel, detected no expression with either technique. We conclude that: (1) intracellular Ca2+ store(s) and store-dependent [Ca2+]i oscillations in human spermatozoa rely primarily on a thapsigargin/cyclopiazonic acid-insensitive Ca2+ pump, which is not a SERCA as characterised in somatic cells; (2) effects of high-dose thapsigargin on spermatozoa primarily reflect non-specific actions on non-SERCAs and; (3) secretory pathway Ca2+ ATPases contribute at least part of this non-SERCA Ca2+ pump activity.

The distribution of the secretory pathway Ca2+ -ATPase (SPCA1) was investigated at both the mRNA and protein level in a variety of tissues. The mRNA and the protein for SPCA1 were relatively abundant in rat brain, testis and testicular derived cells (myoid cells, germ cells, primary Sertoli cells and TM4 cells; a mouse Sertoli cell line) and epididymal fat pads. Lower levels were found in aorta (rat and porcine), heart, liver, lung and kidney. SPCA activities from a number of tissues were measured and shown to be particularly high in brain, aorta, heart, fat pads and testis. As the proportion of SPCA activity compared to total Ca2+ ATPase activity in brain, aorta, fat pads and testis were relatively high, this suggests that SPCA1 plays a major role in Ca2+ storage within these tissues. The subcellular localisation of SPCA1 was shown to be predominantly around the Golgi in both human aortic smooth muscle cells and TM4 cells.