Structural integrity of the cyclin-dependent kinase inhibitor genes, p15, p16 and p18 in myeloid leukaemias

Summary The cyclin-dependent kinase inhibitors known as p15, p16 and p18 have been suggested as candidates for tumour suppressor genes. We examined these genes for their alterations in 46 myeloid leukaemias and 15 myeloid leukaemia cell lines, p16 mRNA expression was studied in 41 myeloid leukaemias. The p15 and p16 genes were either deleted or mutated in myeloid leukaemia lines at a high frequency [6/15 (40%) for p15; 8/15 (53%) for p16] but alterations in primary myeloid leukaemias are much less frequent [2/46 (4%) for p15; 3/46 (6%) for p16]. Alterations of p18 were not found in any of the samples. 13 primary myeloid leukaemia samples had negligible levels of p16 mRNA. In summary, the deletions of p15 and p16 genes identified in the myeloid leukaemia cell lines probably occurred during their in vitro immortalization. Alterations of the pl6 or pl5 gene only occurred in primary acute myeloid leukaemia samples that were of mixed myeloid/ lymphoid lineage (CD19/CD20-positive acute myeloid leukaemia [AML], CD2/CD19-positive AML, and lymphoid blastic crisis of chronic myeloid leukaemia). Further studies are required to determine if the absence of mRNA expression results from inactivation of the p16 gene.     

Analysis of a family of cyclin-dependent kinase inhibitors: p15/MTS2/INK4B, p16/MTS1/INK4A, and p18 genes in acute lymphoblastic leukemia of childhood

A newly recognized family of proteins that inhibit cyclin-dependent kinases (CDKs) termed cyclin-dependent kinase inhibitors (CDKI) have an important role in regulation of cell-cycle progression. A subfamily of these CDKIs (p15INK4B/MTS2, p16INK4/MTS1, and p18) have a high degree of structural and functional homology and are candidate tumor- suppressor genes. We evaluated the mutational status of the p15, p16, and p18 genes in 103 childhood acute lymphoblastic leukemia (ALL) samples and correlated these results with both their clinical data and additional results concerning their loss of heterozygosity in the region of the p15/p16 genes. Homozygous deletions of the p16 gene occurred extremely frequently in T-ALLs (17/22; 77%), and it was also frequent in precursor-B ALLs (12/81; 15%). Homozygous deletions of the p15 gene were also very frequent in T-ALLs (9/22; 41%), and it occurred in 5 of 81 (6%) precursor-B ALL samples. No deletions of p18 was found in any of the 103 ALL samples. Also, no point mutations of the p15, p16, and p18 genes were detected. We correlated p15/p16 alterations at diagnosis with their clinical characteristics as compared with 2,927 other patients treated similarly. Those with p15/p16 alterations were older; had higher white blood cell counts, often with T-cell ALL phenotype; and more frequently had a mediastinal mass at presentation; but they had the same nonremission, relapse, and survival rates at 5 years as did those patients whose blast cells did not have a p15/p16 deletion. To better understand the extent of alterations affecting chromosome 9p21 (location of the p15/p16 genes), loss of heterozygosity (LOH) was examined at D9S171, which is about 1 megabase proximal to the p15/p16 genes. LOH was detected in 15 of 37 (41%) informative samples. Interestingly, of the 24 informative samples that had no detectable alteration of the p15/p16 genes, 7 samples (29%) had LOH at D9S171. In summary, we show in a very large study that p15 and p16, but not p18, CDKI genes are very frequently altered in ALL; those with p15/p16 alterations are more frequently older children, have higher white blood cells at presentation, and often have a T-cell ALL phenotype. The LOH analysis suggests that another tumor-suppressor gene important in ALL also is present on chromosome 9p21.     

Stimulus-induced association of Ca(2+)-binding proteins with the plasma membrane detected in situ by photolabeling of intact chromaffin and PC12 cells.

To investigate the involvement of cytosolic proteins in exocytosis, a system with high temporal and spatial resolution has been developed that allows us to detect the interaction of Ca(2+)- and membrane-binding proteins with the plasma membrane during stimulation of intact chromaffin and PC12 (rat pheochromocytoma) cells. We used 5-iodonaphthalene-1-azide (INA), a hydrophobic label that rapidly partitions into the lipid bilayer of biological membranes. Upon photolysis the label covalently attaches to membrane-embedded domains of proteins. Cells, preincubated with INA in the dark, were stimulated by either 300 microM carbamoylcholine or 60 mM K+ and irradiated (20 s) at various time intervals after stimulation. Subsequently, the cytosolic Ca(2+)- and membrane-binding proteins were isolated in the presence of EGTA (EGTA extract). Of the approximately 40 proteins in the EGTA extract, 15 (15-100 kDa) are labeled in both cell types. Upon stimulation, labeling is increased up to 3-fold in some of the proteins compared to cells labeled under basal conditions. In the absence of external Ca2+, no increase is observed. The rate of label incorporation is similar to the rate of exocytosis in several of these proteins. These results indicate that in the event of triggered exocytosis some of the Ca(2+)-binding proteins interact with the plasma membrane and temporarily embed in the lipid bilayer. Our findings support the hypothesis according to which stimulus-induced alterations in the structure of the Ca(2+)-binding proteins lead to their transient insertion into the membrane and thereby to membrane fusion.     

Renal endothelin receptor type B upregulation in rats with low or high renin hypertension

OBJECTIVES: To evaluate the role of endothelin-1 (ET-1) in hypertension, we investigated density and distribution of ETA and ETB receptors in hearts and kidneys of deoxycorticosterone acetate (DOCA)-salt and 1 kidney -- 1 clip (1K1C) hypertensive rats. METHODS: Five groups of uninephrectomized Wistar rats were put on a low salt diet. Three groups of rats drank tap water and two groups received saline. One group of each regimen received DOCA subcutaneously and two corresponding groups without DOCA served as controls. The fifth group of rats had the renal artery clipped to induce 1K1C hypertension. At 6 weeks, mean arterial pressure (MAP) was recorded and membrane binding assays using 125I-ET-1 were carried out. RESULTS: MAP was increased from control 122 +/- 3 to 155 +/- 6 and 218 +/- 11 mmHg in DOCA-salt and 1K1C rats, respectively, and cardiac weight index was increased. ETA receptors were predominantly expressed in the heart, whereas ETB receptors were predominant in the kidney. In the kidneys, the density of the ETB receptor subtype was upregulated in DOCA-salt and 1K1C rats from 160 +/- 8 to 217 +/- 12 and 190 +/- 2 fmol/mg (P < 0.05), respectively, and ETA tended to be downregulated (P = 0.057). Plasma renin activity was decreased in DOCA-salt rats from 17 +/- 3 to 0.17 +/- 0.01 ng/ml per h and increased in 1K1C rats on low salt diet to 30 +/- 5 ng/ml per h. CONCLUSIONS: Since ETB is the predominant endothelin receptor in the kidneys, upregulation of the ETB receptor mediating vasodilation and downregulation of the ETA receptor mediating vasoconstriction would be compatible with a mainly renal counter-regulatory effect of endothelin-1 to hypertension. Both low and high renin models of hypertension may be affected.     

Influence of Rapid Fluid Loading on Airway Structure and Function in Healthy Humans

BackgroundThe present study examined the influence of rapid intravenous fluid loading (RFL) on airway structure and pulmonary vascular volumes using computed tomography imaging and the subsequent impact on pulmonary function in healthy adults (n = 16).Methods and ResultsTotal lung capacity (ΔTLC = ?6%), forced vital capacity (ΔFVC = ?14%), and peak expiratory flow (ΔPEF = ?19%) decreased, and residual volume (ΔRV = +38%) increased post-RFL (P < .05). Airway luminal cross-sectional area (CSA) decreased at the trachea, and at airway generation 3 (P < .05), wall thickness changed minimally with a tendency for increasing in generation five (P = .13). Baseline pulmonary function was positively associated with airway luminal CSA; however, this relationship deteriorated after RFL. Lung tissue volume and pulmonary vascular volumes increased 28% (P < .001) post-RFL, but did not fully account for the decline in TLC.ConclusionsThese data suggest that RFL results in obstructive/restrictive PF changes that are most likely related to structural changes in smaller airways or changes in extrapulmonary vascular beds.     

Revascularization for chronic critical lower limb ischemia in octogenarians is worthwhile

OBJECTIVE: Advanced age is considered a relative contraindication for surgical revascularization in patients with peripheral arterial occlusive disease. Our aim was to analyze the usefulness of endovascular and surgical revascularization in patients older than 80 years with chronic critical leg ischemia (CLI). Our hypothesis was that the clinical benefit of lower extremity revascularization is limited in octogenarians. METHODS: This was a prospective cohort study with a 1-year follow-up. Subjects included a consecutive series (January 1999 to June 2004) of patients presenting with CLI. Revascularization cohorts were either open surgical or endovascular with conservatively treated patients as a reference group. Prospective follow-up occurred after 30 days and 2, 6, and 12 months. The primary end point was sustained clinical success, defined as a categorical upward shift in clinical symptoms according to Rutherford, without major amputation and without the need for repeated target extremity revascularization (TER). Secondary clinical success was defined accordingly, including repeated TER. Mortality, major amputation, and TER were separately calculated end points. All results were stratified for age categories of nonoctogenarians (<80 years) and octogenarians (> or =80 years). Cumulative outcome was determined by the Kaplan-Meier method, and differences were assessed by log-rank tests. Multivariable analysis was performed by using Cox proportional regression. RESULTS: A total of 376 patients (158 women; mean age, 75.8 +/- 10.7 years) with 416 critically ischemic limbs were analyzed. Overall, 150 patients (39.9%) were older than 80 years, and 85 limbs were treated surgically (26 octogenarians; 30.6%), 207 limbs (96 octogenarians; 46.4%) were treated by endovascular means, and 124 limbs (45 octogenarians; 36.3%) were treated conservatively, including delayed revascularization procedures. Both sustained and secondary clinical success rates, as well as limb salvage rates, were higher in the revascularization cohorts as compared with conservatively treated patients, regardless of age category (P < .001, P < .001, and P = .006, respectively, by Cox proportional hazard model). Mortality was significantly higher in octogenarians (P = .006 by Cox proportional hazard model), particularly within 30 days after surgical revascularization (hazard ratio, 5.35; 95% confidence interval, 1.15-24.9). Patient age category did not affect the rate of major amputations or TER. CONCLUSIONS: Individually tailored revascularization improves the outcome of CLI in octogenarians as well as in nonoctogenarians; even so, endovascular revascularization should be preferred in octogenarians because of the higher mortality associated with surgery.