p73 is over-expressed in vulval cancer principally as the Delta 2 isoform.

p73 was studied in squamous cancers and precursor lesions of the vulva. Over-expression of p73 occurred commonly in both human papillomavirus (HPV)-positive and -negative squamous cell cancers (SCC) and high-grade premalignant lesions. Whereas expression in normal vulval epithelium was detected only in the basal and supra-basal layers, expression in neoplastic epithelium increased with grade of neoplasia, being maximal at both protein and RNA levels in SCC. p73 Delta 2 was the principal over-expressed isoform in the majority of cases of vulval SCC and often the sole form expressed in SCC. Over-expression of p73 was associated with expression of HPV-encoded E7 or with hypermethylation or mutation of p16(INK4a) in HPV-negative cases. There was a close correlation between expression of p73 and p14(ARF) in cancers with loss of p53 function. The frequent over-expression of p73 Delta 2 in neoplastic but not normal vulval epithelium, and its co-ordinate deregulation with other E2F-1 responsive genes suggests a role in the oncogenic process.

Rapid detection of DNA sequence variants by conformation-sensitive capillary electrophoresis.

The identification of novel sequence variants, which may be either disease-causing mutations or silent polymorphisms, in large numbers of samples is becoming the rate-limiting step in associating diseases with specific genes. This is particularly true in light of the imminent arrival of the complete reference sequence of the human genome. A number of techniques have been developed to analyze DNA samples for sequence variants rapidly. We describe a new technique, capillary-based conformation-sensitive gel electrophoresis (capillary CSGE) that transfers mutation detection from acrylamide gel to capillary electrophoresis. Capillary CSGE was able to detect 7/7 short insertion/deletions and 16/22 base substitutions in a series of random single-nucleotide polymorphisms and known variants in the lipoprotein lipase and BRCA2 genes. This technique has the potential to screen many megabases of DNA in a single day.

Sequence variants of the axin gene in breast, colon, and other cancers: an analysis of mutations that interfere with GSK3 binding.

Axin is a recently discovered component of a multiprotein complex containing APC, beta-catenin, GSK3, and PP2A, which functions in the degradation of the beta-catenin protein. As part of WNT signal transduction, the function of the Axin complex is inhibited, leading to the accumulation of beta-catenin. The inappropriate stabilization of beta-catenin has been implicated in a range of human tumors. Two oncogenic mechanisms leading to beta-catenin stabilization are the loss of the APC tumor suppressor protein and the mutational activation of beta-catenin, such that the Axin/APC complex can no longer regulate it. Studies in Drosophila and mammalian tissue culture showed loss of Axin function interfered with beta-catenin turnover and activated beta-catenin/TCF-dependent transcription. Based on these observations, Axin was screened for mutations in a range of human tumor cell lines and primary breast tumor samples. We identified two sequence variants causing amino acid substitutions in four colon cancer cell lines, a Ser-to-Leu at residue 215 in LS513 and a Leu-to-Met at residue 396 in HCT-8, HCT-15, and DLD-1. The Axin L396M mutation was selected for further study since it lay within a region that was shown to interact with glycogen synthase kinase-3. Biochemical and functional studies showed that the L396M change interfered with Axin's ability to bind GSK3. Interestingly, this mutation and a neighboring L392M change differentially altered Axin's ability to interfere with two upstream activators of TCF-dependent transcription, Frat1 and Disheveled.

Spectrum of p53 gene mutations suggests a possible role for ultraviolet radiation in the pathogenesis of advanced cutaneous lymphomas.

There is evidence that the incidence of primary cutaneous lymphoma, like other forms of non-Hodgkin's lymphoma, is increasing, yet little is known of the pathogenetic events involved in this group of disorders. In this study we examine the frequency and spectrum of P53 gene mutations in a large series of primary cutaneous lymphomas, with particular emphasis on tumor stage mycosis fungoides, as it is in these cases that p53 overexpression has previously been reported. Sixty-six samples from 55 patients with primary cutaneous B cell and T cell lymphomas were analyzed for mutations in exons 5-9 of the P53 gene using polymerase chain reaction/single strand conformational polymorphism, and subsequent cloning and sequencing of genomic DNA. Fourteen separate P53 mutations were identified in blood, skin, and lymph node samples in 13 patients (24%). Twelve of 14 mutations occurred at dipyrimidine sites, eight resulting in C-->T transitions and one in a CC-->TT tandem base transition, a mutation spectrum strikingly similar to that reported in nonmelanoma skin cancer and characteristic of DNA damage caused by ultraviolet B radiation. In the subset of patients with mycosis fungoides, P53 mutations were identified in six of 17 patients with tumor-stage but in none of 12 patients with plaque-stage disease (Fisher's exact test p = 0.027). These data suggest a role for ultraviolet radiation in the pathogenesis of primary cutaneous lymphomas and a possible ultraviolet B-related step in the progression of mycosis fungoides from plaque to tumor-stage disease.

A maize pectin methylesterase-like gene, ZmC5, specifically expressed in pollen.

Pectin methylesterase (PME) is responsible for the demethylation of pectin prior to pectin's degradation by the combined activities of polygalacturonase and pectate lyase. We have differentially screened a maize pollen cDNA library to detect cDNA clones whose genes are specifically expressed in pollen. One group of clones resulting from this screen showed similarity (between 18% and 41% identity) with plant and fungal PMEs. The full-length clone from this group, ZmC5, identifies a small gene family (at least 2 members) when used as a probe on genomic Southern blots. Northern analysis reveals that the ZmC5 transcript is expressed specifically in late pollen development. This tissue-specific gene expression programme is further confirmed in transgenic tobacco plants harbouring ZmC5 promoter/GUS chimeric gene constructs.

A transforming p53 mutant, which binds DNA, transactivates and induces apoptosis reveals a nuclear:cytoplasmic shuttling defect.

The DG75 Burkitt lymphoma-derived human B cell line is heterozygous for p53, carrying wild type (WT) and mutant (Arg283His) alleles. The cells constitutively express high levels of both p53 proteins and also Mdm2. Arg283His transactivates the p21Waf1, Mdm2, bax, cyclin G and IGF-BP3 promoters in transient transfection assays equally as well as, if not better than WT p53. It also suppresses the outgrowth of SAOS-2 cells and specifically binds DNA like wild type protein. However, in primary rodent fibroblasts Arg283His fails to suppress transformation by HPV16-E7 and (Ha-)ras and even has modest transforming activity when transfected alone with (Ha-)ras. When Arg283His is transiently transfected into SAOS-2 cells it efficiently induces apoptosis, so - unlike mutants such as Arg175Pro - its behaviour in transformation assays does not clearly correlate with loss of the apoptosis function. Immunofluorescence staining of both REF transformants and transiently transfected SAOS-2 revealed that this unusual mutant becomes excluded from the nucleus and produces striking cytoplasmic fluorescence. The best correlation with transformation, therefore, appears to be the lack of nuclear retention of Arg283His. Since this mutation does not map to any known nuclear localization signal and its presence seems to result in aberrant exclusion from the nucleus, then it may prove very useful in exploring mechanisms involved in the nuclear:cytoplasmic shuttling of p53.

cDNA cloning of a third human C2-domain-containing class II phosphoinositide 3-kinase, PI3K-C2gamma, and chromosomal assignment of this gene (PIK3C2G) to 12p12.

Phosphoinositide (PI) 3-kinases have been shown to have critical roles in signaling pathways that regulate proliferation, oncogenic transformation, cell survival, cell migration, and intracellular protein trafficking. We have previously used reverse-transcription polymerase chain reaction methods to identify novel PI 3-kinase isoforms in normal human breast and in lymph nodes containing metastatic breast cancer. Here we report the cDNA cloning of a Class II PI 3-kinase found in normal breast tissue. This gene (PIK3C2G) encodes the third distinct protein of the human Class II PI 3-kinase family, PI3K-C2gamma. PIK3C2G was mapped to chromosome 12 at 12p12 by fluorescence in situ hybridization.

Identification of four novel human phosphoinositide 3-kinases defines a multi-isoform subfamily.

Phosphoinositide (PI) 3-kinases have critical roles in diverse cellular signalling processes and in protein trafficking. This suggests that like other intracellular signalling molecules, e.g., phospholipase C and protein kinase C, there might be a large family of PI 3-kinase isoforms with the individual members having discrete signalling roles. Reverse transcription-polymerase chain reaction methods, using degenerate oligonucleotide primers against the lipid kinase consensus region, revealed eight sequences from human cDNA containing a high degree of identity to the family of PI 3-kinases. The sequences obtained included the previously described p110 alpha, p110 beta, and p110 gamma isoforms and HsVps34. Additionally, we have identified four novel sequences which are related to PI 3-kinases. Three of the novel sequences would appear to form a distinct sub-family of PI 3-kinases. We report the expression of these novel PI 3-kinases in human tissues and in cells derived from normal breast.

p53 mutations implicate sunlight in post-transplant skin cancer irrespective of human papillomavirus status.

Mutations in p53 were detected in 11/23 (48%) of non melanoma skin cancers in renal allograft recipients and in 5/8 (63%) of sporadic tumours from immune competent patients. 9/12 (75%) of mutations in transplant patients and all 5 mutations in non transplant tumours were consistent with damage caused by ultraviolet (u.v.) irradiation. DNA sequences, predominantly of the epidermodysplasia verruciformis (EV) subgroup, were detected in 9/23 (39%) of transplant tumours and in 2/8 (25%) of eight non-transplant tumours. There was no relationship between HPV status and p53 mutation, HPV DNA being present in 5/16 (31%) of tumours with p53 mutation and 6/15 (40%) of tumours lacking p53 mutation. These data are consistent with an important role for sunlight in the development of post-transplant skin cancer, and with limited functional data suggesting that E6 proteins of the cutaneous and EV-related papillomaviruses do not target p53 for ubiquitin-mediated degradation.

Coexpression of H2-Mb and H2-Ab genes during fetal and postnatal development.

The major histocompatibility complex (MHC) class II-like molecules, H2-M, have an essential role in processing and presentation of antigens by the MHC class II molecules, because functional inactivation of these genes lead to surface expression of MHC class II molecules devoid of associated foreign peptides. We have used in situ hybridization to examine the expression of MHC class II and H2-Mb genes in embryonic and neonatal mice and show here that expression of H2-Ab and H2-Mb mRNA is absent in 13.5-day-old mice. However, mRNA for both genes could be detected in the thymus of 14.5- and 15.5-day-old embryos, and the patterns of hybridization suggested that the two genes were expressed in the same cells. In spleen and thymus of neonatal mice the H2-Ab and H2-Mb genes were also coexpressed, with expression being localized to the white pulp of the spleen and to the thymic medulla, which are rich in antigen-presenting cells. The steady-state levels of H2-Ab mRNA appeared to be approximately 10 to 14 times greater that the level of H2-Mb mRNA molecules, irrespectively of the tissue and age, reflecting the different functions of the two molecules.

Membrane healing and restoration of contractility after mechanical injury in isolated skeletal muscle fibers of the frog.

In single isolated skeletal muscle fibers of the frog, we studied (i) the recovery from large sarcolemmal mechanical injuries of the response to electric stimulation and (ii) the integrity of the sarcolemma under the light microscope. In Ringer's solution, the damaged cells stopped contracting and deteriorated completely within 1 hr. In the presence of phosphatidylcholine (0.025 g/ml in Ringer's solution), the injured cells initially responded with local twitches. Within 0.5 hr, contractility and membrane integrity started to recover and both were back to control levels within 3 hr. When these cells were placed back in normal Ringer's solution, they remained viable and active for several hours. Our results suggest that phosphatidylcholine can protect muscle fibers from the effects of sarcolemmal injury.

Effects of thapsigargin in normal and pretreated with ryanodine guinea pig cardiomyocytes.

We compared the effects of thapsigargin (TG), a selective blocker of Ca(2+)-adenosinetriphosphatase of sarcoplasmic reticulum (SR), and ryanodine (Ry) in the single isolated myocytes of guinea pig ventricular myocardium loaded with indo 1 acetoxymethyl ester (AM). TG (2 x 10(-7) M) inhibited the rapid phase of Ca2+ transient, increased time to peak intracellular Ca2+ concentration ([Ca2+]i) from 158 +/- 12 to 391 +/- 60 ms and decreased the total amplitude of the transient to 89 +/- 4% of the pre-TG control. Time to peak of contractions increased from 350 +/- 47 to 410 +/- 37 ms and total duration from 666 +/- 62 to 850 +/- 198 ms. Total amplitude of contractions was hardly affected. In the cells not loaded with indo 1-AM TG decreased the amplitude of contractions to 71 +/- 3% of control. When the effects of TG were fully developed, the cells ceased to respond to 1 s of superfusion with 15.0 mM caffeine with transient elevation of [Ca2+]i and/or transient contracture. TG did not affect the amplitude or time course of Ca2+ current (ICa) or the current-voltage relation. We propose that Ca2+ transients and contractions in the cells treated with TG were initiated by sarcolemmal Ca2+ influx. Ry (1.0 microM) initiated similar changes in the time course of Ca2+ transients and contractions as TG; however, total amplitude of the transients and contractions was reduced to 78 +/- 5 and 55 +/- 7% of the control, respectively. The SR Ca2+ was also depleted by Ry. TG superfused over the cells pretreated with Ry increased the amplitude of Ca2+ transients and respective contractions to the pre-Ry level. TG did not affect the ICa in the cells pretreated with Ry nor did it change configuration of action potentials to increase the Ca2+ influx. We propose that the effect of Ry on amplitude of Ca2+ transients and contractions results from the trapping of a fraction of sarcolemmal Ca2+ influx by the SR and its rapid release into subsarcolemmal space. From there it is extruded out of the cell by Na(+)-Ca2+ exchange before ever reaching the contractile system.

Non-homogeneous Ca release in isolated frog skeletal muscle fibres.

We have examined the spatial distribution of [Ca2+]i during tetanic stimulation in frog skeletal muscle cells using a fluorescence imaging method. We have found a completely unexpected pattern of Ca release: Ca is released forming gradients composed of spots of very significant and slow fluctuations of calcium release. Our findings could be explained if the calcium release process in skeletal muscle is influenced significantly by [Ca2+]i, such as in cardiac muscle, and suggests that the SR/Ca release control can include the established voltage-dependent plus a cardiac-like process of calcium-induced Ca release and a Ca release inhibition by Ca.

Differential activation of myofibrils during fatigue in phasic skeletal muscle cells.

In fatigued muscles the T-system is swollen; thus the action potential may fail to travel along the T-system or the T-tubule terminal cisternae signal may fail to bring about TC Ca2+ release. This would lead to a decrease in the number of myofibrils activated and in force development, but if fatigue is the result of a generalized process, all the myofibrils would be affected equally leading to a lower activation of all of them. We have investigated this possibility in isolated twitch muscle fibres by giving them repetitive tetanic stimulations until fatigue developed. The behaviour of myofibrils was followed with cinemicrophotography. Before fatigue, no lack of shortening of myofibrils could be found. During fatigue groups of myofibrils became wavy. When exposed to caffeine, the wavy myofibrils disappeared and tension similar to the control developed. The tension-caffeine concentration relationship was shifted to the left after development of fatigue. In low Na+ solution fatigue developed faster and after reintroducing normal Ringer, tension recovered substantially. K-contractures were smaller during fatigue. These results indicate that in this type of fatigue, a step in the EC coupling chain of events is involved in its development.

Fourier analysis of the nuclear and cytoplasmic shapes of living two-cell murine embryos.

Living two-cell mouse embryos were flushed out from the oviduct 17, 24 and 36 hours after fertilization in order to obtain cells in the G1S, early G2 and late G2 phases of the second cell cycle. The nuclei of the living cells were stained with Hoechst 33342. The coordinates of the contour shapes of the entire cells (cellular contours) were registered by contour image processing with a TV camera coupled with a computer; the contours of the nuclei were computed by means of a digitizer coupled with the computer. Fourier analysis of the cellular and nuclear contours revealed systematic modifications in the folding of the cells and nuclei in the course of the murine second cell cycle. The progression of cells through the second cell cycle was correlated with an increasing diversification of cellular and nuclear shape, with the diversification being much more pronounced in the nuclear shapes.

Heterogeneity of cytoplasmic and nuclear shape of lymphocytes during progression of chronic lymphocytic leukemia.

Peripheral blood from ten healthy subjects and from 44 patients at stages 0, I, II, III, IV of chronic lymphocytic leukemia (CLL), type B, was routinely smeared, fixed and stained by the May-Grunwald-Giemsa method. Fourier analysis of nuclear and cytoplasmic shape of smeared lymphocytes was carried out for the range 1-20 of harmonics (describing the pattern of contour folding in quantitative terms). In addition the roughness coefficients (describing the summarized measure of contour folding of an individual cell) were calculated and computer evaluated. Cytoplasmic contour shape of smeared lymphocytes in the 6-10 harmonic range discriminates well between lymphocytes of healthy subjects and those of each CLL stage. This discrimination was the result of richer folding of CLL lymphocytes. Nuclear contour shape of lymphocytes in the 6-10 harmonic range fails to discriminate between lymphocytes of healthy subjects and those of CLL, but it discriminates well between lymphocytes of various stages of CLL, with the exception of stages I/II and III/IV. When Fourier analysis was carried out on lymphocytes of combined stages I + II and III + IV, the shape differences were even more accentuated. We conclude that nuclear and cytoplasmic contour shape is a phenotypic feature of lymphocytes that is markedly modified in the course of CLL progression; this feature may be used as a new parameter in CLL.