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PubMed1997

Transforming growth factor-beta receptor type I and type II expression during murine hair follicle development and cycling.

Although the TGF-beta family of growth factors probably regulates skin and hair follicle development, its exact role is still quite ill-defined. Here, we characterize the correlative expression pattern of the interdependent high affinity receptor proteins for TGF-beta1 and TGF-beta3, TGF-beta receptor type I (TGF-betaRI) and TGF-beta receptor type II (TGF-betaRII), during hair follicle development and cycling in C57BL/6 mice. During neonatal follicle development, TGF-betaRII immunoreactivity is confined to epithelial cells. Focal epidermal TGF-betaRII expression is seen even before actual hair placode formation. In contrast to the TGF-betaRII immunoreactivity in the outer root sheath, precortical hair matrix and inner root sheath cells were TGF-betaRII negative during hair bulb morphogenesis. TGF-betaRI (Alk-5) immunoreactivity largely overlapped the TGF-betaRII expression pattern, but was more widespread. During hair follicle cycling in adolescent mice, TGF-betaRII immunoreactivity was restricted to follicles, and was strikingly hair cycle dependent (maximal immunoreactivity: anagen VI and early catagen). Again, TGF-betaRI (Alk-5) immunoreactivity co-localized with TGF-betaRII immunoreactivity, but was more extensive. Reverse transcriptase polymerase chain reaction analysis of TGF-betaRII mRNA confirmed peak transcript levels in back skin with most hair follicles in the anagen VI-catagen transformation. mRNA levels of TGF-betaRI (Alk-5) did not vary significantly during the hair cycle, whereas those of TGF-betaRI (threonine-serine kinase 7 L) declined during early anagen, and were maximal during the anagen-catagen transition. This provides a basis for defining the choreography of TGF-beta-related signalling during hair follicle morphogenesis and cycling, introduces intraepidermal TGF-betaRII immunoreactivity as a marker for imminent follicle development, and supports the concept that both TGF-betaRII and TGF-betaRI stimulation is involved in, but not restricted to, the control of catagen induction.

Aging↗
PubMed1996

Diradylglycerol formation in cholecystokinin-stimulated rabbit pancreatic acini. Assessment of precursor phospholipids by means of molecular species analysis.

The aim of the present study was to assess the origin of the 1,2-diradylglycerols produced during prolonged hormonal stimulation of rabbit pancreatic acini by comparison of their relative molecular species composition with that of the major acinar phospholipids. Both phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) consisted of 1,2-diacyl as well as 1-alk-1-2-acyl species. In contrast, phosphatidylinositol (PtdIns), phosphatidylserine and phosphatidic acid existed only in the 1,2-diacyl form. Acinar cells did not contain detectable amounts of 1-alkyl-2-acyl phospholipids. Similarly, the acinar 1,2-diradylglycerol fraction consisted of 1,2-diacylglycerols and 1-alk-1-enyl-2-acylglycerols. Mass 1,2-diradylglycerol measurements revealed that prolonged stimulation with cholecystokinin resulted in a marked and sustained increase in acinar 1,2-diradylglycerol content. Based on the relative amounts of the 1,2-diacyl species present in both the 1,2-diradylglycerol fraction and the individual phospholipids, it is calculated that under control conditions 60% of the 1,2-diacylglycerols originate from PtdCho and 40% from PtdIns, whereas under stimulatory conditions 53% is calculated to be derived from PtdCho, 46% from PtdIns and 1% from PtdEtn. Likewise, it is calculated that in control as well as stimulated acini 100% of the 1-alk-l-enyl-2-acylglycerols originate from plasmenylcholine. Further evidence in favour of the idea that at least a considerable part of the 1,2-diacylglycerols produced during prolonged hormonal stimulation originate from inositolphospholipids is provided by the observation that labeling of phosphatidylinositol 4,5-bisphosphate with inorganic phosphate reached isotopic equilibrium markedly faster under stimulatory conditions as compared to the control situation, which is in agreement with an elevated turnover rate. The data presented support the idea that PtdCho and inositolphospholipids are the major precursors in basal and stimulated 1,2-diradylglycerol production in rabbit pancreatic acini.

Animals↗
PubMed2005

Non-mediastinal grey zone lymphomas and report from the workshop.

Grey zone lymphomas represent borderline lesions between classical Hodgkin lymphoma (cHL) and other morphologically and immunophenotypically related diseases and entities like nodular lymphocyte predominant HL, T-cell rich B-cell lymphoma, ALK-negative anaplastic large cell lymphoma (ALCL), diffuse large B cell lymphoma (DLBCL) anaplastic variant and primary mediastinal LBCL. The sharp definition of morphological, immunophenotypical and molecular features of the "text-book cases" of each disease and the comparison with grey zone cases has reduced most of the latter cases. Two reports in this workshop dealt with the problematic non-mediastinal grey zone lymphomas, one with a cHL of T cell-type presenting in the skin as a ALK-negative ALCL and the other with the grey zone between cHL of B-cell type and ALK-negative ALCL.

Education↗
PubMed1998

RNAse protection assay for BMP type I receptors.

Bone morphogenetic proteins (BMPs) signal through a receptor complex comprising two distinct transmembrane serine/threonine kinases referred to as type I and type II receptors. The identification of at least three classes of type I receptors (ALK-2, -3, -6) and three classes of type II receptors for the BMPs, raises the possibility of multiple receptor complexes which may have distinct functions. An RNAse protection assay has been established which allows simultaneous analyses of the mRNAs for ALK-2, -3, and -6. As a test system, cultures of pluripotent C3H10T1/2 cell line were incubated in the presence or absence of BMP-7, and the relative levels of the receptor mRNAs were determined. Changes in receptor mRNA levels for ALK-2 and -3 were detected during the proliferation phase and adipogenic differentiation of the pluripotent C3H10T1/2 cell line.

Adipocytes↗
PubMed2005

Influence of pre-exercise acidosis and alkalosis on the kinetics of acid-base recovery following intense exercise.

The purpose of this study was to measure the recovery kinetics of pH and lactate for the conditions of pre-exercise acidosis, alkalosis, and placebo states. Twelve trained male cyclists completed 3 exercise trials (110% workload at VO2max), ingesting either 0.3 g/kg of NH4Cl (ACD), 0.2 g/kg of Na+HCO3- and 0.2 g/kg of sodium citrate (ALK), or a placebo (calcium carbonate) (PLAC). Blood samples (heated dorsal hand vein) were drawn before, during, and after exercise. Exercise-induced acidosis was more severe in the ACD and PLAC trials (7.15 +/- 0.06, 7.21 +/- 0.07, 7.16 +/- 0.06, P < 0.05, for ACD, ALK, PLAC, respectively). Recovery kinetics for blood pH and lactate, as assessed by the monoexponential slope constant, were not different between trials (0.057 +/- 0.01, 0.050 +/- 0.01, 0.080 +/- 0.02, for ACD, ALK, PLAC, respectively). Complete recovery of blood pH from metabolic acidosis can take longer than 45 min. Such a recovery profile is nonlinear, with 50% recovery occurring in approximately 12 min. Complete recovery of blood lactate can take longer than 60 min, with 50% recovery occurring in approximately 30 min. Induced alkalosis decreases metabolic acidosis and improves pH recovery compared to acidodic and placebo conditions. Although blood pH and lactate are highly correlated during recovery from acidosis, they recover at significantly different rates.

Acid-Base Equilibrium↗
PubMed2002

Gene cloning and characterization of multiple alkane hydroxylase systems in Rhodococcus strains Q15 and NRRL B-16531.

The alkane hydroxylase systems of two Rhodococcus strains (NRRL B-16531 and Q15, isolated from different geographical locations) were characterized. Both organisms contained at least four alkane monooxygenase gene homologs (alkB1, alkB2, alkB3, and alkB4). In both strains, the alkB1 and alkB2 homologs were part of alk gene clusters, each encoding two rubredoxins (rubA1 and rubA2; rubA3 and rubA4), a putative TetR transcriptional regulatory protein (alkU1; alkU2), and, in the alkB1 cluster, a rubredoxin reductase (rubB). The alkB3 and alkB4 homologs were found as separate genes which were not part of alk gene clusters. Functional heterologous expression of some of the rhodococcal alk genes (alkB2, rubA2, and rubA4 [NRRL B-16531]; alkB2 and rubB [Q15]) was achieved in Escherichia coli and Pseudomonas expression systems. Pseudomonas recombinants containing rhodococcal alkB2 were able to mineralize and grow on C(12) to C(16) n-alkanes. All rhodococcal alkane monooxygenases possessed the highly conserved eight-histidine motif, including two apparent alkane monooxygenase signature motifs (LQRH[S/A]DHH and NYXEHYG[L/M]), and the six hydrophobic membrane-spanning regions found in all alkane monooxygenases related to the Pseudomonas putida GPo1 alkane monooxygenase. The presence of multiple alkane hydroxylases in the two rhodococcal strains is reminiscent of other multiple-degradative-enzyme systems reported in Rhodococcus.

Amino Acid Sequence↗
PubMed1980

Treponema innocens lipids and further description of an unusual galactolipid of Treponema hyodysenteriae.

The lipids of Treponema innocens, type strain B256, formerly considered a nonpathogenic isolate of T. hyodysenteriae, have been analyzed and compared with the lipids of T. hyodysenteriae. The lipids of T. innocens comprised 16% of the cell dry weight. Polar lipids amounted to about two-thirds of the total lipids and consisted of 61.9% phospholipids and 38.1% glycolipid. Neutral lipids consisted mainly of sterols. The phospholipids were principally phosphatidylglycerol, phosphatidylcholine, and cardiolipin. Minor amounts of lysophosphatidylcholine, sphingomoyelin, and a relatively nonpolar, unidentified phospholipid were present. The latter lipid has not been detected in T. hyodysenteriae. The glycolipid fraction of T. innocens contained a single component, monoglucosyldiglyceride, in contrast to the occurrence in T. hyodysenteriae of two components: monogallactosyldiglyceride and a less-polar glycolipid tentatively identified as acylmonogalactosyldiglyceride (the additional acyl moieties being 86.6% acetyl, 11.6% propionyl, and 1.6% n-butyryl groups). Alk-1-enyl ether analogs comprised 24.6% of the total phospholipids and glycolipid of T. innocens, or about one-third of the amount in T. hyodysenteriae. The acyl and alk-1-enyl moieties of T. innocens consisted of greater than or equal to 92% of 14:0, iso-15:0, and 16:0 chains. In contrast to T. hyodysenteriae, anteiso-15:0 moieties were not detected, and a reversed distribution of 14:0 and iso-15:0 alk-1-enyl moieties occurred in the two species.

Galactolipids↗
PubMed1995

Growth on octane alters the membrane lipid fatty acids of Pseudomonas oleovorans due to the induction of alkB and synthesis of octanol.

Growth of Pseudomonas oleovorans GPo1, which contains the OCT plasmid, on octane results in changes in the membrane phospholipid fatty acid composition. These changes were not found for GPo12, an OCT-plasmid-cured variant of GPo1, during growth in the presence or absence of octane, implying the involvement of OCT-plasmid-encoded functions. When recombinant strain GPo12(pGEc47) carrying the alk genes from the OCT plasmid was grown on octane, the cells showed the same changes in fatty acid composition as those found for GPo1, indicating that such changes result from induction and expression of the alk genes. This finding was corroborated by inducing GPo12(pGEc47) with dicyclopropylketone (DCPK), a gratuitous inducer of the alk genes. Further experiments showed that the increase of the mean acyl chain length of fatty acids is related to the expression of alkB, which encodes a major integral membrane protein, while the formation of trans unsaturated fatty acids mainly results from the effects of 1-octanol, an octane oxidation product.

1-Octanol↗
PubMed2002

Rubredoxins involved in alkane oxidation.

Rubredoxins (Rds) are essential electron transfer components of bacterial membrane-bound alkane hydroxylase systems. Several Rd genes associated with alkane hydroxylase or Rd reductase genes were cloned from gram-positive and gram-negative organisms able to grow on n-alkanes (Alk-Rds). Complementation tests in an Escherichia coli recombinant containing all Pseudomonas putida GPo1 genes necessary for growth on alkanes except Rd 2 (AlkG) and sequence comparisons showed that the Alk-Rds can be divided in AlkG1- and AlkG2-type Rds. All alkane-degrading strains contain AlkG2-type Rds, which are able to replace the GPo1 Rd 2 in n-octane hydroxylation. Most strains also contain AlkG1-type Rds, which do not complement the deletion mutant but are highly conserved among gram-positive and gram-negative bacteria. Common to most Rds are the two iron-binding CXXCG motifs. All Alk-Rds possess four negatively charged residues that are not conserved in other Rds. The AlkG1-type Rds can be distinguished from the AlkG2-type Rds by the insertion of an arginine downstream of the second CXXCG motif. In addition, the glycines in the two CXXCG motifs are usually replaced by other amino acids. Mutagenesis of residues conserved in either the AlkG1- or the AlkG2-type Rds, but not between both types, shows that AlkG1 is unable to transfer electrons to the alkane hydroxylase mainly due to the insertion of the arginine, whereas the exchange of the glycines in the two CXXCG motifs only has a limited effect.

Alkanes↗
PubMed1999

A cytotoxic phenotype does not predict clinical outcome in anaplastic large cell lymphomas.

AIM: To investigate whether anaplastic large cell lymphomas (ALCL) expressing cytotoxic proteins have a relatively worse clinical outcome compared with ALCL lacking a cytotoxic phenotype. METHODS: 59 primary cases of ALCL originating from different sites were investigated by immunohistochemistry for the presence of the cytotoxic proteins T cell intracytoplasmic antigen (TIA-1) and granzyme B in the neoplastic cells. Since site of origin and expression of anaplastic lymphoma kinase (ALK) strongly influence prognosis, the presence of a cytotoxic phenotype was also investigated in relation to the primary site of origin (lymph node, gut, or skin) and ALK expression. The prognostic value was investigated by analysis of overall and relapse-free survival time, including Cox regression analysis. RESULTS: 39 of 59 ALCL (66%) appeared to have a cytotoxic phenotype as shown by expression of TIA-1 or granzyme B or both in the neoplastic cells. The presence of a cytotoxic phenotype did not have any influence on prognosis. Even when the survival data were corrected for site of origin and stage at presentation or were analysed separately for ALK positive and negative cases, no prognostic influence of a cytotoxic phenotype was observed. CONCLUSIONS: In primary biopsies of patients with ALCL, the presence of a cytotoxic phenotype is not related to clinical outcome of the disease.

Adult↗
PubMed2005

A new locus for hereditary haemorrhagic telangiectasia (HHT3) maps to chromosome 5.

Patients with hereditary haemorrhagic telangiectasia (HHT, or Osler-Weber-Rendu syndrome) have variable presentation patterns and a high risk of preventable complications. Diagnostic tests for mutations in endoglin (HHT type 1) and ALK-1 (HHT type 2) are available. Some HHT patients are now known to have HHT-juvenile polyposis overlap syndrome due to Smad4 mutations. Families were ascertained following the presentation of probands for embolization of pulmonary arteriovenous malformations. Genome-wide linkage studies using over 700 polymorphic markers, and sequencing of candidate genes, were performed. In a previously described HHT family unlinked to endoglin or ALK-1, linkage to Smad4 was excluded, and no mutations were identified in the endoglin, ALK-1, or Smad4 genes. Two point LOD scores and recombination mapping identified a 5.4 cM HHT3 disease gene interval on chromosome 5 in which a single haplotype was inherited by all affected members of the pedigree. The remainder of the genome was excluded to a 2-5 cM resolution. We are currently studying a further family potentially linked to HHT3. We conclude that classical HHT with pulmonary involvement can result from mutations in an unidentified gene on chromosome 5. Identification of HHT3 should further illuminate HHT pathogenic mechanisms in which aberrant transforming growth factor (TGF)-beta signalling is implicated.

Chromosome Mapping↗
PubMed2003

Visceral manifestations in hereditary haemorrhagic telangiectasia type 2.

Hereditary haemorrhagic telangiectasia (HHT) is a genetic vascular disorder characterised by epistaxis, telangiectases, and visceral manifestations. The two known disease types, HHT1 and HHT2, are caused by mutations in the endoglin (ENG) and ALK-1 genes, respectively. A higher frequency of pulmonary arteriovenous malformations (AVMs) has been reported for HHT1 while HHT2 is thought to be associated with a lower penetrance and milder disease manifestations. In this study, we present 10 families with an ALK-1 genotype. Visceral manifestations were detected in 24 (26%) of the 93 HHT2 patients from nine of the families and included gastrointestinal bleeding (14%), intrahepatic shunts (6%), and AVMs in the lung (4%) and brain (3%). Gastrointestinal bleeding, the most frequent visceral manifestation, was reported in six of the 10 families, mostly in patients over the age of 50. These patients also had frequent epistaxis and suffered from anaemia, often requiring blood transfusions. The identification of ALK-1 mutations in subjects with a suspected diagnosis and without clinical signs of HHT argue in favour of a molecular diagnosis. We also analysed the data published on 44 families with HHT2 and conclude that visceral manifestations occur in 26 of these families and affect 30% of HHT2 patients. This is considered an underestimate given incomplete and variable screening for lung, brain, and/or liver involvement in different clinical centres. These findings, however, stress the need for an early diagnosis of HHT that can be useful for the early control of associated visceral involvement.

Activin Receptors, Type I↗
PubMed1984

Resolution of alkenylacylglycerol moieties of natural glycerophospholipids by gas-liquid chromatography on polar capillary columns.

A sensitive and practical procedure has been developed for the determination of the sn-1-alk-1-enyl-2-acylglycerol moieties of natural glycerophospholipids using capillary gas-liquid chromatography with flame ionization detection. The method gives excellent resolution and quantitative estimates for molecular species according to carbon number and degree of unsaturation. The diradylglycerols are released from the glycerophospholipids by hydrolysis with phospholipase C and the alk-1-enylacylglycerols are resolved by thin-layer chromatography. Either the trimethylsilyl or the tertiary-butyldimethylsilyl ethers are then resolved at 250 degrees C by gas-liquid chromatography on a glass capillary column wall-coated with a polar cyanopropylsiloxane polymer using hydrogen as the carrier gas. The method has been applied to the analysis of the alk-1-enylacyl- and diacyl-glycerol moieties of the phosphatidylethanolamines of rat heart and kidney.

Animals↗
PubMed1990

Detoxification of secondary products of lipid peroxidation in the cytosol of a mouse fibroblast cell line.

A cytosolic fraction prepared from a mouse fibroblast cell line reduced secondary products of lipid peroxidation, including alkenals, alkanals, alk-2-enals, and alka-2,4-dienals, using NADH or NADPH. An alternative route for detoxification of alk-2-enals and alka-2,4-dienals in the cell line cytosol was via glutathione conjugation. The major glutathione transferase in the cell line cytosol was partially characterized by glutathione-agarose chromatography and chromatofocusing. High glutathione peroxidase activity suggested that the enzyme was an alpha family glutathione transferase. The major glutathione transferase also efficiently catalyzed the conjugation of alk-2-enals and alka-2,4-dienals with glutathione.

Animals↗
PubMed2006

Hyperoxia modulates TGF-beta/BMP signaling in a mouse model of bronchopulmonary dysplasia.

Prematurely born infants who require oxygen therapy often develop bronchopulmonary dysplasia (BPD), a debilitating disorder characterized by pronounced alveolar hypoplasia. Hyperoxic injury is believed to disrupt critical signaling pathways that direct lung development, causing BPD. We investigated the effects of normobaric hyperoxia on transforming growth factor (TGF)-beta and bone morphogenetic protein (BMP) signaling in neonatal C57BL/6J mice exposed to 21% or 85% O(2) between postnatal days P1 and P28. Growth and respiratory compliance were significantly impaired in pups exposed to 85% O(2), and these pups also exhibited a pronounced arrest of alveolarization, accompanied by dysregulated expression and localization of both receptor (ALK-1, ALK-3, ALK-6, and the TGF-beta type II receptor) and Smad (Smads 1, 3, and 4) proteins. TGF-beta signaling was potentiated, whereas BMP signaling was impaired both in the lungs of pups exposed to 85% O(2) as well as in MLE-12 mouse lung epithelial cells and NIH/3T3 and primary lung fibroblasts cultured in 85% O(2). After exposure to 85% O(2), primary alveolar type II cells were more susceptible to TGF-beta-induced apoptosis, whereas primary pulmonary artery smooth muscle cells were unaffected. Exposure of primary lung fibroblasts to 85% O(2) significantly enhanced the TGF-beta-stimulated production of the alpha(1) subunit of type I collagen (Ialpha(1)), tissue inhibitor of metalloproteinase-1, tropoelastin, and tenascin-C. These data demonstrated that hyperoxia significantly affects TGF-beta/BMP signaling in the lung, including processes central to septation and, hence, alveolarization. The amenability of these pathways to genetic and pharmacological manipulation may provide alternative avenues for the management of BPD.

Animals↗
PubMed2003

Alterations in TGF-beta1 expression in lambs with increased pulmonary blood flow and pulmonary hypertension.

The mechanisms responsible for pulmonary vascular remodeling in congenital heart disease with increased pulmonary blood flow remain unclear. We developed a lamb model of congenital heart disease and increased pulmonary blood flow utilizing an in utero placed aortopulmonary vascular graft (shunted lambs). Morphometric analysis of barium-injected pulmonary arteries indicated that by 4 wk of age, shunts had twice the pulmonary arterial density of controls (P < 0.05), and their pulmonary vessels showed increased muscularization and medial thickness at both 4 and 8 wk of age (P < 0.05). To determine the potential role of TGF-beta1 in this vascular remodeling, we investigated vascular changes in expression and localization of TGF-beta1 and its receptors TbetaRI, ALK-1, and TbetaRII in lungs of shunted and control lambs at 1 day and 1, 4, and 8 wk of life. Western blots demonstrated that TGF-beta1 and ALK-1 expression was elevated in shunts compared with control at 1 and 4 wk of age (P < 0.05). In contrast, the antiangiogenic signaling receptor TbetaRI was decreased at 4 wk of age (P < 0.05). Immunohistochemistry demonstrated shunts had increased TGF-beta1 and TbetaRI expression in smooth muscle layer and increased TGF-beta1 and ALK-1 in endothelium of small pulmonary arteries at 1 and 4 wk of age. Moreover, TbetaRI expression was significantly reduced in endothelium of pulmonary arteries in the shunt at 1 and 4 wk. Our data suggest that increased pulmonary blood flow dysregulates TGF-beta1 signaling, producing imbalance between pro- and antiangiogenic signaling that may be important in vascular remodeling in shunted lambs.

Activin Receptors, Type I↗
PubMed2006

Effects of chronic NaHCO3 ingestion during interval training on changes to muscle buffer capacity, metabolism, and short-term endurance performance.

This study determined the effects of altering the H(+) concentration during interval training, by ingesting NaHCO(3) (Alk-T) or a placebo (Pla-T), on changes in muscle buffer capacity (beta m), endurance performance, and muscle metabolites. Pre- and posttraining peak O(2) uptake (V(O2 peak)), lactate threshold (LT), and time to fatigue at 100% pretraining V(O2 peak) intensity were assessed in 16 recreationally active women. Subjects were matched on the LT, were randomly placed into the Alk-T (n = 8) or Pla-T (n = 8) groups, and performed 8 wk (3 days/wk) of six to twelve 2-min cycle intervals at 140-170% of their LT, ingesting NaHCO(3) or a placebo before each training session (work matched between groups). Both groups had improvements in beta m (19 vs. 9%; P < 0.05) and V(O2 peak) (22 vs. 17%; P < 0.05) after the training period, with no differences between groups. There was a significant correlation between pretraining beta m and percent change in beta m (r = -0.70, P < 0.05). There were greater improvements in both the LT (26 vs. 15%; P = 0.05) and time to fatigue (164 vs. 123%; P = 0.05) after Alk-T, compared with Pla-T. There were no changes to pre- or postexercise ATP, phosphocreatine, creatine, and intracellular lactate concentrations, or pH(i) after training. Our findings suggest that training intensity, rather than the accumulation of H(+) during training, may be more important to improvements in beta m. The group ingesting NaHCO(3) before each training session had larger improvements in the LT and endurance performance, possibly because of a reduced metabolic acidosis during training and a greater improvement in muscle oxidative capacity.

Adaptation, Physiological↗
PubMed2004

Transcriptional activation of p21(waf1/cip1) by alkylphospholipids: role of the mitogen-activated protein kinase pathway in the transactivation of the human p21(waf1/cip1) promoter by Sp1.

Alkylphospholipids (ALKs) are a novel class of antitumor agents with an unknown mechanism of action. The first ALK tested in the clinic, miltefosine, has been approved recently in Europe for the local treatment of patients with cutaneous metastasis. Perifosine, the only available oral ALK, is being studied currently in human cancer clinical trials. We have shown previously that perifosine induces p21(waf1/cip1) in a p53-independent fashion and that induction of p21(waf1/cip1) is required for the perifosine-induced cell cycle arrest because cell lines lacking p21(waf1/cip1) are refractory to perifosine. In this report, we investigated the mechanism by which perifosine induces p21(waf1/cip1) protein expression. We observed that perifosine induces the accumulation of p21(waf1/cip1) mRNA without affecting p21(waf1/cip1) mRNA stability. Using several p21(waf1/cip1) promoter-driven luciferase reporter plasmids, we observed that perifosine activates the 2.4-kb full-length p21(waf1/cip1) promoter as well as a p21 promoter construct lacking p53-binding sites, suggesting that perifosine activates the p21(waf1/cip1) promoter independent of p53. The minimal p21 promoter region required for perifosine-induced p21 promoter activation contains four consensus Sp1-binding sites. Mutations in each particular Sp1 site block perifosine-induced p21(waf1/cip1) expression. Moreover, we showed that perifosine activates the mitogen-activated protein/extracellular signal-regulated kinase pathway, and this activation promotes the phosphorylation of Sp1 in known mitogen-activated protein kinase residues (threonine 453 and 739), thereby leading to increased Sp1 binding and enhanced p21(waf1/cip1) transcription. These results represent a novel mechanism by which alkylphospholipids modulate transcription, and may contribute to the discovery of new signal transduction pathways crucial for normal and neoplastic cell cycle control.

Alkylation↗