Association of single nucleotide polymorphisms in endothelin family genes with the progression of atherosclerosis in patients with essential hypertension.

Endothelin-1 (ET-1) is a potent vasoconstrictive peptide and its activity is mediated by the receptors ET type A (EDNRA) and ET type B (EDNRB). Although ET-1 is thought to play an important role in the development of atherosclerosis, it remains unclear whether polymorphisms of ET-1 family genes, including the ET-1 gene (EDN1), EDNRA, EDNRB and the genes for endothelin converting enzymes 1 and 2 (ECE1 and ECE2), are associated with the progression of atherosclerosis. We investigated the relationship between 11 single nucleotide polymorphisms (SNPs) of ET-1 family genes (including three in EDN1, one in EDNRA, two in EDNRB, four in ECE1 and one in ECE2) and atherosclerotic changes assessed using pulse wave velocity (PWV) and carotid ultrasonography in 630 patients with essential hypertension (EHT). In male subjects, we found significant differences in brachial-ankle PWV (baPWV) in additive and recessive models in EDNRB-rs5351 after Bonferroni correction. Also in male subjects, there were significant differences in mean intima-media thickness (IMT) in additive and recessive models in EDNRA-rs5333 after Bonferroni correction. We found no significant correlation between any SNPs in the ET family genes and baPWV, IMT and Plaque score (PS) in female subjects. Furthermore, after multiple logistic regression analysis, only EDNRB-rs5351 indicated as an independent risk of atherosclerosis in male hypertensive subjects. Of the endothelin-related genes, EDNRB-rs5351 was the most susceptible SNP associated with atherosclerosis in male hypertensives, and the genetic background may be involved in the progression of atherosclerosis in EHT patients.

Prevalence of Anderson-Fabry disease in male patients with late onset hypertrophic cardiomyopathy.

BACKGROUND: Although studies have suggested that "late-onset" hypertrophic cardiomyopathy (HCM) may be caused by sarcomeric protein gene mutations, the cause of HCM in the majority of patients is unknown. This study determined the prevalence of a potentially treatable cause of hypertrophy, Anderson-Fabry disease, in a HCM referral population. METHODS AND RESULTS: Plasma alpha-galactosidase A (alpha-Gal) was measured in 79 men with HCM who were diagnosed at > or =40 years of age (52.9+/-7.7 years; range, 40-71 years) and in 74 men who were diagnosed at <40 years (25.9+/-9.2 years; range, 8-39 years). Five patients (6.3%) with late-onset disease and 1 patient (1.4%) diagnosed at <40 years had low alpha-Gal activity. Of these 6 patients, 3 had angina, 4 were in New York Heart Association class 2, 5 had palpitations, and 2 had a history of syncope. Hypertrophy was concentric in 5 patients and asymmetric in 1 patient. One patient had left ventricular outflow tract obstruction. All patients with low alpha-Gal activity had alpha-Gal gene mutations. CONCLUSION: Anderson-Fabry disease should be considered in all cases of unexplained hypertrophy. Its recognition is important given the advent of specific replacement enzyme therapy.

Apolipoprotein J/clusterin is induced in vascular smooth muscle cells after vascular injury.

BACKGROUND: Understanding the precise molecular mechanisms underlying the phenomenon of restenosis after PTCA may help us to develop a new strategy for the treatment of restenosis after PTCA. The purpose of this study was to identify the genes involved in vascular restenosis. METHODS AND RESULTS: Applying a differential hybridization method to a model of the balloon-injured rabbit aorta, we identified 6 cDNA clones that were upregulated after injury. Northern blot showed that 5 genes, but not apolipoprotein J (apoJ)/clusterin, were constitutively expressed in noninjured aorta and upregulated after balloon injury. ApoJ mRNA was not detectable in noninjured aorta (control), began to be expressed at 6 hours after injury, showed a peak level at 24 hours (a 48-fold increase), gradually declined, and returned to the control level at 24 weeks. Western blot and immunohistochemistry demonstrated no expression of apoJ protein in noninjured aorta, an expression of apoJ at 2 days after balloon injury, and a peak level (a 55-fold increase) at 2 to 8 weeks. The expression of apoJ protein continued until 24 weeks after injury. In situ hybridization revealed that apoJ mRNA was expressed in smooth muscle cells (SMCs) of media at 2 days after injury and in SMCs of media and neointima at 2 weeks. To analyze the function of apoJ, stably transfected rabbit SMCs were created. The expression of apoJ stimulated proliferation and migration of SMCs. CONCLUSIONS: ApoJ is dramatically induced in media and neointima after vascular injury, suggesting that apoJ contributes to restenosis after angioplasty.

PIP2 and ATP cooperatively prevent cytosolic Ca2+-induced modification of ATP-sensitive K+ channels in rat pancreatic beta-cells.

The factors that influence functional coupling between the sulfonylurea receptor (SUR1) and Kir6.2 subunits of ATP-sensitive K+ (K+(ATP)) channels were studied in rat pancreatic beta-cells using patch clamp and microfluorometric techniques. Tolbutamide at 10 micromol/l inhibited K+(ATP) channels in association with occurrence of action currents, but further exposure of beta-cells to the drug for 30 min or longer resulted in reappearance of K+(ATP) channel events. Half-maximal inhibition concentration (IC50) for tolbutamide was 1.5 microl/mol in 2.8 mmol/l glucose, and it was increased to 13.3 micromol/l when the cellular metabolism was inhibited by 0.5 mmol/l 2,4-dinitrophenol (DNP) for 5 min. Tolbutamide at 10 micromol/l induced an increase in cytosolic Ca2+ concentration ([Ca2+]i), and its amplitude was markedly reduced following exposure to 0.5 mmol/l DNP or long-term (30 min) exposure to 10 micromol/l tolbutamide. This tolbutamide insensitivity, as assessed by the [Ca2+]i response, was not observed when the external Ca2+ was omitted during the long-term exposure to tolbutamide. In cell-attached membrane patches, the tolbutamide insensitivity was also produced by treatment of cells with 150 micromol/l diazoxide and 25 mmol/l KCl in the presence, but not absence, of 2 mmol/l Ca2+ in the external solution. When the cytoplasmic face of inside-out membrane patches was treated with higher Ca2+ concentrations (2 micromol/l), both ADP-evoked activation and tolbutamide-induced inhibition of K+ ATP channels were attenuated with retaining ATP-induced inhibition, indicating the modification of K+(ATP) channels. The Ca2+-induced channel modification was prevented partially by phosphatidylinositol 4,5-bisphosphate (PIP2) and completely by ATP and PIP2 together, but not by ATP alone. Treatment of the channel with cytochalasin D, a disrupter of F-actin, evoked channel modification similar to that induced by Ca2+. The modification was prevented completely by phalloidin, a stabilizer of F-actin. In conclusion, long-term exposure to tolbutamide or metabolic inhibition causes modification of K+ ATP channels via mechanisms involving Ca2+-dependent reaction. The modification, which may reflect functional disconnection between SUR1 and Kir6.2, is prevented by ATP and PIP2, which may act cooperatively to stabilize membrane cytoskeletons (F-actin structures).

Interventricular septal and free wall dynamics in hypertrophic cardiomyopathy.

Two-dimensional echocardiography was used to analyze interventricular septal and free wall dynamics in eight normal subjects and eight patients with hypertrophic cardiomyopathy. Upper, middle and lower septal and corresponding free wall motion and thickening were analyzed using both fixed and floating reference systems. The lower and midseptal dynamics did not seem to differ significantly between the two groups and the lower septum seemed to move more than the corresponding free wall (probability [p] less than 0.05). The upper septum moved and thickened less than the rest of the septum in both groups (p less than 0.05), but was less dynamic in patients with hypertrophic cardiomyopathy than in normal subjects when the fixed reference system was used (p less than 0.05). It is concluded that the interventricular septum in hypertrophic cardiomyopathy is not akinetic. Previously reported hypokinesia of the septum in hypertrophic cardiomyopathy may be due to sampling of the upper septum by M-mode echocardiography and to the fixed system of reference used by M-mode echocardiography.

Atrial systolic notch on the interventricular septal echogram: an echocardiographic sign of constrictive pericarditis.

Interventricular septal motion during ventricular diastole was analyzed using M-mode echocardiography in 13 patients with constrictive pericarditis and 12 patients with restrictive cardiomyopathy. In seven of eight patients with constrictive pericarditis in sinus rhythm, an abnormal "atrial systolic" notch was observed consisting of abrupt initial posterior motion toward the left ventricle approximately at the middle of the P wave and subsequent anterior motion at the end of the P wave and termination before the R wave. This notch was absent during atrial premature beats that were recorded in two patients. The atrial systolic notch was not observed in any patient with restrictive cardiomyopathy. The septal notch in early ventricular diastole previously described in constrictive pericarditis was seen in 62% of patients with constrictive pericarditis and 25% of patients with restrictive cardiomyopathy. Thus, an abnormal atrial systolic notch may be an additional useful sign to differentiate constrictive pericarditis from restrictive cardiomyopathy. The mechanism may be related to transient late diastolic pressure gradients between both ventricles resulting from asynchrony of left and right atrial contractions.

Angled interventricular septum on echocardiography: anatomic anomaly or technical artifact?

An acutely angled interventricular septum has been reported to constitute a distinct two-dimensional echocardiographic geometric pattern that may permit a false M-mode echocardiographic recording of asymmetric septal hypertrophy. In light of experience suggesting that the angle between the aortic root and interventricular septum varied with the intercostal space of the transducer, 45 subjects were prospectively studied by two-dimensional and M-mode echocardiography. Parasternal long- and short-axis views were obtained from two to four intercostal spaces in each subject. Two-dimensional echographic cursor-generated M-mode echocardiograms were obtained from the long-axis views; interventricular septal and left ventricular posterior wall thickness was measured from both the two-dimensional and M-mode echocardiograms. On two-dimensional echocardiography, the angle between the aortic root and septum became more acute as a progressively lower intercostal space was used (p less than 0.001). Although no change in septal thickness was apparent, the septal thickness significantly increased as a progressively lower intercostal space was used. On M-mode echocardiography, 21 subjects (47%) demonstrated asymmetric septal hypertrophy (septal/posterior wall thickness ratio greater than 1.3) from at least one intercostal space, but this was confirmed by the two-dimensional technique in only 4 subjects (9%). Thus, a two-dimensional echocardiographic recording of an angled interventricular septum can be produced by positioning the transducer in a low intercostal space, and caution must be used in the interpretation of asymmetric septal hypertrophy on M-mode echocardiograms. Two-dimensional echocardiography is a useful means of identifying subjects with apparent asymmetric septal hypertrophy that often may be the result of a technical artifact.

Hyperemic response of intracoronary contrast agents during two-dimensional echographic delineation of regional myocardium.

Several intracoronary echo contrast agents that provided satisfactory regional myocardial delineation with two-dimensional echocardiography were compared in 15 dogs and their effects on coronary blood flow were examined. Reproducible delineation of myocardium subserved from the intracoronary echo contrast injection site was achieved with hand-agitated agents containing greater than or equal to 30% Renografin, greater than or equal to 30% glucose, greater than or equal to 30% sucrose or 6% dextran. After a 2 cc injection of the echo contrast agent, peak hyperemic augmentation of coronary flow was 56.7 +/- 54.4% for 6% dextran, 116.0 +/- 71.1% for 30% Renografin, 119.3 +/- 47.8% for 30% sucrose, 173.8 +/- 38.3% for 30% glucose. Although, 6% dextran resulted in the lowest and shortest hyperemic response of the four agents, computer-derived echo contrast appearance-disappearance analysis indicated a prolonged myocardial contrast decay half-life (21.0 seconds). On the other hand, 30% Renografin had a more rapid myocardial echo contrast washout (T 1/2 = 15.5 seconds), but a significantly greater hyperemic effect was observed. It is concluded that development of echo contrast agents for myocardial contrast two-dimensional echocardiographic assessment of myocardial perfusion will require consideration of alterations in coronary flow due to contrast-induced hyperemia.

Clinical improvement in hypertrophic cardiomyopathy after inferior myocardial infarction.

In cases of hypertrophic cardiomyopathy, the pathophysiologic role of the systolic pressure gradient across the left ventricular outflow tract is the subject of continued controversy. A patient with this disorder is described whose symptoms and provokable intraventricular gradient disappeared after inferior myocardial infarction. Diastolic left ventricular pressures were essentially unchanged, the isovolumic relaxation period became prolonged and the ejection fraction decreased from 0.77 to 0.61 after infarction. The peak ejection rate was unchanged, but the disappearance of systolic anterior motion of the mitral valve leaflet and obstructive manifestations may have resulted from enlarged mid to late systolic ventricular volumes. This case suggests a direct relation between symptoms and intraventricular pressure gradient in certain patients with hypertrophic cardiomyopathy.

Doppler index combining systolic and diastolic myocardial performance: clinical value in cardiac amyloidosis.

OBJECTIVES: This study was designed to determine the clinical value of a Doppler-derived index of combined systolic and diastolic myocardial performance in the assessment of cardiac amyloidosis. BACKGROUND: Cardiac amyloidosis is an infiltrative disease with diastolic and systolic dysfunction. Therefore, the index of myocardial performance combining systolic and diastolic time intervals could be a useful predictor of clinical outcome in cardiac amyloidosis. METHODS: The study included 45 patients with biopsy-proved amyloidosis and 45 age-matched normal subjects. All patients had typical echocardiographic features of amyloid cardiac involvement. A Doppler-derived index, defined as the sum of isovolumetric contraction time and isovolumetric relaxation time divided by ejection time, was measured from left ventricular outflow and mitral inflow Doppler velocity profiles recorded during routine echocardiography. The index as well as conventional systolic or diastolic echocardiographic/Doppler variables were related to subsequent outcome. RESULTS: The isovolumetric contraction and relaxation times were prolonged and ejection time was shortened (p < 0.001) in patients with amyloidosis compared with that in normal subjects, resulting in a marked increase of the index from normal values (p < 0.001). In the amyloid group the index was highest in patients with a low stroke index or with both shortened mitral deceleration time and lower ejection fraction. By univariate analysis, New York Heart Association functional class, the index, ejection fraction and mitral deceleration time were significant predictors of outcome. However, by multivariate stepwise regression analysis, functional class and the index were the only independent predictors of survival. CONCLUSIONS: The Doppler-derived index of combined systolic and diastolic myocardial performance correlates with global cardiac dysfunction and is a useful predictor of clinical outcome in patients with cardiac amyloidosis.

Diagnosis of coronary stenosis by two-dimensional echographic study of dysfunction of ventricular segments during and immediately after pacing.

The adequacy of two-dimensional echocardiography during right atrial pacing for the detection and characterization of coronary artery stenosis was examined in 10 closed chest dogs. Pacing at successively higher rates up to 210 beats/min was carried out in the control state and again during a 70% left anterior descending coronary artery stenosis-induced with intracoronary plugs. Left ventricular short-axis echographic cross sections were obtained at several levels of the left ventricle. After computer-aided standardized subdivision, contractile function of the global section and its subsegments was characterized by computed systolic fractional area change percent and wall thickening percent. Ventricular segments supplied from the site of the 70% coronary stenosis were delineated in a low papillary level cross section by a myocardial contrast echographic technique, and these segments demonstrated significant dysfunction during pacing at 150 to 210 beats/min. Echographic observation of the involved segments immediately after pacing revealed a maximal depression of function 5 seconds after pacing, equivalent to dysfunction at peak pacing, with function returning to control levels within about 2 minutes. Both maximal pacing and early postpacing studies facilitated satisfactory discrimination of ischemic from normally perfused myocardial segments. These experiments show that right atrial pacing study with quantitative two-dimensional echocardiography may serve to detect and assess a coronary stenosis associated with minor or no cardiac dysfunction in the rest state.

Atypical echocardiographic and angiographic presentation of a postoperative pseudoaneurysm of the left ventricle after repair of a true aneurysm.

Subsequent to the repair of a true aneurysm from the posteromedial-basal aspect of the left ventricle, a 58 year old man developed a draining wound at the site of the sternotomy. Two-dimensional echocardiography revealed recurrence of the aneurysm at the site of the previous aneurysm repair. This aneurysm had a wide neck and looked similar in appearance to the previous true aneurysm. However, at surgery the patient was found to have a ventricular pseudoaneurysm with a cardiocutaneous fistula.

Verification of myocardial contrast two-dimensional echocardiographic assessment of perfusion defects in ischemic myocardium.

Myocardial contrast two-dimensional echocardiography was used in 21 closed chest dogs to assess its ability to delineate the extent of underperfused acutely ischemic myocardium. An agitated saline-Renografin echocardiographic contrast agent was injected into the left main coronary artery after left anterior descending coronary artery occlusion, and the size of the contrast echo-free area characterizing the perfusion defect was outlined in short-axis cross sections of the left ventricle. In 13 dogs, monastral blue dye was injected after 45 minutes of coronary artery occlusion and before sacrifice to provide anatomic delineation of underperfused zones in equivalent sections. Perfusion defects assessed by contrast two-dimensional echocardiography correlated well with those delineated by monastral blue dye (r = 0.91). Contrast echocardiographic study was also performed in eight other dogs at 5 hours of occlusion, after which infarct size was measured with triphenyl-tetrazolium-chloride. Contrast echocardiographic outline of the perfusion deficiency correlated but slightly overestimated the extent of necrosis (r = 0.88). It is concluded that contrast two-dimensional echocardiography can detect and outline the underperfused "risk area" during acute coronary artery occlusion, and may also permit assessment of the extent of myocardial infarction.

Two-dimensional contrast echocardiography. I. In vitro development and quantitative analysis of echo contrast agents.

To facilitate the passage of echo contrast agents through the microcirculation and the echocardiographic study of myocardial perfusion, ultrasonic energy (sonication) was employed to produce contrast agents consisting of relatively uniform, stable and small (less than 10 mu diameter) gaseous microbubbles suspended in liquid solutions. The size and persistence of the microbubbles was verified by light microscopy and an in vitro system were employed for comparative assessment of peak echo amplitude and echo persistence characteristics of various contrast agents. The study indicated that although a variety of hand-agitated and sonicated contrast agents provided satisfactory echo intensities, sonication was clearly superior to the hand-agitation method, because sonication produced smaller, more uniform and more stable microbubbles that may be suitable for myocardial contrast echocardiography. It is concluded that of the contrast agents examined, sonicated solutions of sorbitol (70%) and dextrose (70%) appeared to have particular potential because of the small sizes of the microbubbles (6 +/- 2 and 8 +/- 3 mu, respectively) and their prolonged in vitro persistence. The use of sonication to produce standardized, small and stable microbubbles should facilitate physiologic passage of the contrast agent through the capillary beds and allow two-dimensional imaging of the left heart myocardium during right-sided, aortic root, coronary sinus or intracoronary contrast injections.

Correlation of myocardial echo contrast disappearance rate ("washout") and severity of experimental coronary stenosis.

The relation between experimental coronary stenosis and myocardial contrast echo disappearance rate ("washout") was investigated in anesthetized closed chest dogs. Of 13 dogs, 8 had serial contrast echographic studies with two successive degrees of coronary stenosis (50 and 70%) produced by threading stenotic plugs into the proximal left circumflex coronary artery. Studies were repeated with complete coronary occlusion achieved by inflation of an intracoronary balloon immediately proximal to the plugs. Myocardial contrast echograms were recorded in short-axis cross sections of the left ventricle after intracoronary injection of 2 ml hand-agitated saline-Renografin solution through a catheter placed in the coronary artery. An echo contrast washout index (t 1/2) was measured by digital processing computer analysis of successive end-diastolic images obtained by two-dimensional echocardiography during myocardial contrast agent injection. The injection to injection correlation coefficient of these t 1/2 measurements was satisfactory (r = 0.87, standard error of estimate 4.8 seconds). Involved segment t 1/2 measurements were found to be significantly altered by intracoronary stenosis and occlusion, ranging from 23 +/- 6 seconds (mean +/- standard deviation) in the control state, 29 +/- 9 and 44 +/- 10 seconds for 50 and 70% stenosis, respectively, and 104 +/- 35 seconds for total occlusion. It was concluded that myocardial contrast two-dimensional echocardiographic measurement of t 1/2 appears to be a useful index of the degree of coronary stenosis.

Pericardial thickness measured with transesophageal echocardiography: feasibility and potential clinical usefulness.

OBJECTIVES: This study assessed the reliability of transesophageal echocardiographic measurements of pericardial thickness and the potential diagnostic usefulness of this technique. BACKGROUND: Transthoracic echocardiography cannot reliably detect thickened pericardium. The superior resolution achieved with transesophageal echocardiography should allow better pericardial definition. METHODS: Pericardial thickness measured at 26 locations in 11 patients with constrictive pericarditis who underwent intraoperative transesophageal echocardiography was compared with pericardial thickness measured with electron beam computed tomography. Intraobserver and interobserver variabilities were determined. Pericardial thickness was then measured in 21 normal subjects. With these values as a guide, two observers reviewed 37 transesophageal echocardiographic studies to determine whether echocardiographic measurement of pericardial thickness could be used to distinguish diseased from normal pericardium. RESULTS: The correlation between echocardiographic and computed tomographic measurements (r > or = 0.95, SE < or = 0.06 mm, p < 0.0001) was excellent. The +/-2 SD limits of agreement were +/-1.0 mm or less for pericardial thickness < 5.5 mm and +/-2.0 mm or less for the entire range of thicknesses. Intraobserver and interobserver agreements were good. Mean normal pericardial thickness was 1.2 +/- 0.8 mm (+/-2 SD) and did not exceed 2.5 mm. Pericardial thickness > or = 3 mm on transesophageal echocardiography was 95% sensitive and 86% specific for the detection of thickened pericardium. CONCLUSIONS: Measurement of pericardial thickness with transesophageal echocardiography is reproducible and should be a valuable adjunct in assessing constrictive pericarditis.

Repeated thermal therapy improves impaired vascular endothelial function in patients with coronary risk factors.

OBJECTIVES: We sought to determine whether sauna therapy, a thermal vasodilation therapy, improves endothelial function in patients with coronary risk factors such as hypercholesterolemia, hypertension, diabetes mellitus and smoking. BACKGROUND: Exposure to heat is widely used as a traditional therapy in many different cultures. We have recently found that repeated sauna therapy improves endothelial and cardiac function in patients with chronic heart failure. METHODS: Twenty-five men with at least one coronary risk factor (risk group: 38 +/- 7 years) and 10 healthy men without coronary risk factors (control group: 35 +/- 8 years) were enrolled. Patients in the risk group were treated with a 60 degrees C far infrared-ray dry sauna bath for 15 min and then kept in a bed covered with blankets for 30 min once a day for two weeks. To assess endothelial function, brachial artery diameter was measured at rest, during reactive hyperemia (flow-mediated endothelium-dependent dilation [%FMD]), again at rest and after sublingual nitroglycerin administration (endothelium-independent vasodilation [%NTG]) using high-resolution ultrasound. RESULTS: The %FMD was significantly impaired in the risk group compared with the control group (4.0 +/- 1.7% vs. 8.2 +/- 2.7%, p < 0.0001), while %NTG was similar (18.7 +/- 4.2% vs. 20.4 +/- 5.1%). Two weeks of sauna therapy significantly improved %FMD in the risk group (4.0 +/- 1.7% to 5.8 +/- 1.3%, p < 0.001). In contrast, %NTG did not change after two weeks of sauna therapy (18.7 +/- 4.2% to 18.1 +/- 4.1%). CONCLUSIONS: Repeated sauna treatment improves impaired vascular endothelial function in the setting of coronary risk factors, suggesting a therapeutic role for sauna treatment in patients with risk factors for atherosclerosis.

M-mode echocardiography in constrictive pericarditis.

M-mode echocardiograms from 40 patients with proven constrictive pericarditis and 40 subjects without evidence of cardiac disease were reviewed for features previously described in constrictive pericarditis. In this large series, no single feature of the M-mode echocardiogram could be considered diagnostic, although a pattern of normal left ventricular size and systolic function, mild left atrial dilation, flattened diastolic left ventricular posterior wall motion and abnormal septal motion was found in most patients. It is concluded that the M-mode echocardiogram can provide findings suggestive of constrictive pericarditis but must be used in conjunction with hemodynamic and other studies to establish the diagnosis.

Alteration of p16 (CDKN2) gene is associated with interleukin-2-induced tumor cell growth in adult T-cell leukemia.

Because tumorigenesis frequently involves the dysfunction of cell cycle-related proteins, we examined the effect of mutations in CDK inhibitor p16 and its linked genomic loci p15, cl.B, and 1063.7 on the growth of primary adult T-cell leukemia (ATL) cells. Southern blot analysis of primary ATL cells showed a significantly higher incidence of p16 gene alteration in acute ATL than in chronic ATL [67.7% (23/34) vs. 26.1% (6/23), respectively; p<0.003]. Similarly, polymerase chain reaction (PCR) analysis of p16 exon 2 revealed a higher incidence of alteration in acute ATL than in chronic ATL [52.9% (18/34) vs. 26.1% (6/23), respectively; p<0.05]. PCR-single strand conformation polymorphism analysis of exons 1 and 2 of p16 showed no mutations in the patients, with normal pattern by Southern blotting or PCR analysis. Notably five of six chronic ATL patients with abnormal p16 genes progressed to acute crisis within 4 months. PCR analysis of the p16 linked loci 1063.7, p15 exon 2, and cl.B found homozygous deletion in 55.9%, 20.6%, and 2.9% of acute ATL cells and 39.1%, 13.0%, and 0% of chronic ATL cells, respectively, showing no relationship of homozygous deletion in either loci with disease subtypes. In most cases, deletions were seen in multiple genes, including p16. Acute ATL cells had a higher frequency of multigene deletions than chronic ATL cells [44.1% vs. 17.4%; p<0.05]. When leukemic cells were analyzed for interleukin 2 (IL-2) responsive growth, only p16 gene alteration was directly associated with leukemic cell growth activity. Among leukemic cells showing high IL-2 responsiveness, 73.1% (19/26) had p16 gene alteration vs. 27.8% (5/18) of leukemic cells that showed low IL-2 responsiveness (p<0.005). p16 gene alteration was found in 73.3% (14/19) of leukemic cells showing high autonomous growth rates but in only 40.0% (10/25) of those leukemic cells showing low autonomous growth (p<0.03). These results suggest the following: alteration of p16-related genomic regions in ATL is usually a wide rearrangement including the p16 gene; within this region, only p16 gene alteration is associated with disease aggressiveness; and p16 gene deletion may be a proximate event in leukemogenesis.

NF-kappaB involvement in the activation of primary adult T-cell leukemia cells and its clinical implications.

The HTLV-I provirus-encoded Tax protein induces NF-kappaB in Tax-transfected Jurkat T cells or HTLVL-I- infected T cells in vitro. Tax induction of NF-kappaB is presumed to be involved in proliferation and activation of primary leukemia cells in vivo. Recent studies have demonstrated that NF-kappaB activities in human T cells are mediated by at least four c-Rel-related DNA binding proteins - p50, p55, p75 and p85. We examined the significance of NF-kappaB induction in primary adult T cell leukemia cells and the induction kinetics of each of the four NF-kappaB species. Marked NF-kappaB activity was detected using an electrophoretic mobility shift assay (EMSA) in the primary cells of patients with acute disease, but little activity was noted in the cells of chronic patients. NF-kappaB activity was enhanced in a time-dependent manner in acute type cells cultured with mitogen-free medium; there was no induction of activity in chronic type cells. UV crosslinking demonstrated all four species of NFkappaB complex - high levels of p50 and lower levels of p55 and p75, in acute type cells; chronic type cells showed only the p50. As a control, normal resting T cells similarly showed only p50; control cells showed little change in activity when cultured without mitogenic stimulation, analogous to chronic type ATL. Northern blotting revealed enhancement of c-rel (encoding p85) and KBFI (encoding p50 and p55) expression in acute type cells during culture, while there was no significant enhancement of mRNAs in chronic type ATL cells or unstimulated normal T cells. Northern blotting also revealed that Tax is upregulated at the mRNA level in acute- but not chronic-type cells during culture. Expression of c-rel and KBF1 mRNAs in acute type cells appeared to be related to Tax mRNA expression. These results suggest that Tax is capable of inducing nuclear expression of all four NF-kappaB species in primary ATL cells of acute type patients, with marked effects on p55, p75, and p85. Tax induction of NF-kappaB species is regulated, at least in part, at a pretranslational level involving increases in c-rel and KBF1 mRNA.