Small-molecule RETRA suppresses mutant p53-bearing cancer cells through a p73-dependent salvage pathway.

Identification of unique features of cancer cells is important for defining specific and efficient therapeutic targets. Mutant p53 is present in nearly half of all cancer cases, forming a promising target for pharmacological reactivation. In addition to being defective for the tumor-suppressor function, mutant p53 contributes to malignancy by blocking a p53 family member p73. Here, we describe a small-molecule RETRA that activates a set of p53-regulated genes and specifically suppresses mutant p53-bearing tumor cells in vitro and in mouse xenografts. Although the effect is strictly limited to the cells expressing mutant p53, it is abrogated by inhibition with RNAi to p73. Treatment of mutant p53-expressing cancer cells with RETRA results in a substantial increase in the expression level of p73, and a release of p73 from the blocking complex with mutant p53, which produces tumor-suppressor effects similar to the functional reactivation of p53. RETRA is active against tumor cells expressing a variety of p53 mutants and does not affect normal cells. The results validate the mutant p53-p73 complex as a promising and highly specific potential target for cancer therapy.

DAP-kinase participates in TNF-alpha- and Fas-induced apoptosis and its function requires the death domain.

Death-associated protein (DAP)-kinase is a calcium/calmodulin regulated serine/threonine kinase that carries ankyrin repeats, a death domain, and is localized to the cytoskeleton. Here, we report that this kinase is involved in tumor necrosis factor (TNF)-alpha and Fas-induced apoptosis. Expression of DAP-kinase antisense RNA protected cells from killing by anti-Fas/APO-1 agonistic antibodies. Deletion of the death domain abrogated the apoptotic functions of the kinase, thus, documenting for the first time the importance of this protein domain. Overexpression of a fragment encompassing the death domain of DAP-kinase acted as a specific dominant negative mutant that protected cells from TNF-alpha, Fas, and FADD/MORT1-induced cell death. DAP-kinase apoptotic function was blocked by bcl-2 as well as by crmA and p35 inhibitors of caspases, but not by the dominant negative mutants of FADD/MORT1 or of caspase 8. Thus, it functions downstream to the receptor complex and upstream to other caspases. The multidomain structure of this serine/threonine kinase, combined with its involvement in cell death induced by several different triggers, place DAP-kinase at one of the central molecular pathways leading to apoptosis.

Expression of the normal p53 gene induces differentiation of K562 cells.

The multistep nature of human cancers is well illustrated by chronic myelogenous leukemia (CML), a clonal hematologic malignancy with two distinct phases: chronic and acute. Transition between these phases is characterized by unregulated growth and loss of differentiation of myeloid cells and their progenitors. We recently reported that loss of normal p53 expression correlates with transition from the chronic to acute phase in at least 25% of cases of CML. However, the precise relationship between this loss and biologic features of acute-phase CML is uncertain. To study this question, we artificially expressed normal p53 in K562, an erythroid acute-phase CML cell line lacking normal p53 expression. Biological effects were assessed by determining several growth parameters and by measuring synthesis of hemoglobin, a feature of mature erythroid cells. K562 cells expressing normal p53 had an increased proportion of cells in G1 versus S + G2, a longer doubling time and a lower growth saturation density than control K562 cells or K562 cells with antisense p53. Cells with normal p53 also expressed up to 50-fold more hemoglobin than controls. These data are consistent with the notion that loss of p53 expression may be responsible for many of the features of acute-phase CML cells. The data also demonstrate direct involvement of p53 in differentiation processes.

DAP-kinase loss of expression in various carcinoma and B-cell lymphoma cell lines: possible implications for role as tumor suppressor gene.

DAP-kinase is a novel calmodulin dependent serine/threonine kinase that carries ankyrin repeats and the death domain. It was recently isolated, by a functional selection approach of gene cloning, as a positive mediator of programmed cell death. In this study the expression of DAP-kinase was examined in the cell lines derived from various human neoplasms. DAP-kinase mRNA and protein expression were below the limit of detection in eight out of ten neoplastic derived B-cell lines. In six out of 14 examined bladder carcinoma, in three out of five renal cell carcinoma, and in four out of ten tested breast carcinoma cell lines, the DAP-kinase protein levels were below detection limits or lower than 1% compared to the positive cell lines. Interestingly, DAP-kinase expression could be restored in some of the negative bladder carcinoma and B-cell lines by treatment of cells with 5'-azadeoxycytidine that causes DNA demethylation. The high frequency of loss of DAP-kinase expression in human tumor cell lines, and the occasional involvement of methylation in this process raise the possibility that this novel mediator of cell death may function as a tumor suppressor gene.

Stress-induced secretion of growth inhibitors: a novel tumor suppressor function of p53.

p53 tumor suppressor gene controls cell response to a variety of stresses inducing growth arrest or apoptosis in damaged cells. It largely determines the sensitivity of tumor and normal cells to radiation and chemotherapy, and, therefore, defines both the efficacy and limitations of anti-cancer treatment. To determine molecular mechanisms of p53-dependent stress response in normal tissues we identified and compared the spectra of radiation-responsive genes in cells of different origin and p53 status using a cDNA array hybridization technique. The majority of genes identified were p53-dependent and cell type specific. Several of the new p53 responders encode known secreted growth inhibitory factors. This suggests that p53, in addition to its intrinsic antiproliferation activity, can cause 'bystander effect' by inducing export of growth suppressive stimuli from damaged cells to neighboring cells. Consistently, a p53-dependent accumulation of factors, which causes growth inhibitory effects in a variety of cell lines, was found after gamma irradiation in the media from established and primary cell cultures and in the urine of irradiated mice. Moreover, p53-dependent factors released by normal human fibroblasts potentiated the cytotoxic effect of a chemotherapeutic drug on co-cultivated tumor cells. This suggests a previously unknown role for normal cells in chemo- and radiation therapy of cancer.

Upregulation of Meis1 and HoxA9 in acute lymphocytic leukemias with the t(4 : 11) abnormality.

Rearrangements of the human ALL-1 gene are frequently encountered in acute lymphocytic leukemias (ALL) and acute myeloid leukemias (AML). These rearrangements are mostly due to chromosome translocations and result in production of chimeric proteins composed of the N-terminal fragment of ALL-1 and the C-terminal segments of the partner proteins. The most common chromosome translocation involving ALL-1 is the t(4 : 11) associated with ALL. ALL-1 is the human homologue of Drosophila trithorax and directly activates transcription of multiple Hox genes. A preliminary DNA microarray screen indicated that the Meis1, HoxA9 and AC133 genes were overexpressed in ALLs with t(4 : 11), compared to ALLs with very similar phenotype but without the chromosomal abnormality. These genes, as well as additional five Hox genes, were subjected to comprehensive semi-quantitative or quantitative RT-PCR analysis in 57 primary ALL and AML tumors. Meis1 and HoxA9 were found expressed in 13/14 of ALLs with the t(4 : 11) and in 8/8 of AMLs with ALL-1 rearrangements. The two genes were not consistently transcribed in other types of ALL. AC133 was transcribed in 13/14 of ALLs with t(4 : 11), but in only 4/8 of AMLs with ALL-1 rearrangements. HoxA10 was expressed in most leukemias with ALL-1 alterations, but was also transcribed in PrePreB CD10(-) ALLs lacking the t(4 : 11). Expression of HoxA5, HoxA7, HoxC8 and HoxC10 did not correlate with ALL-1 rearrangements. Coexpression of Meis1 and HoxA9, overexpression of HoxA10, and overexpression or fusion of HoxA9 were previously implicated in certain acute myeloid leukemias in mice and humans. The present work suggests that upregulation of Meis1, HoxA9, and possibly HoxA10 might also play a role in pathogenesis of acute lymphocytic and acute myeloid leukemias associated with ALL-1 fusions.

Initiation and progression of chronic myelogenous leukemia.

Different aspects related to initiation of chronic myelogenous leukemia by the t(9;22) translocation and progression of the disease were investigated. Computer search indicated that the repeat within BCR exon I has significant sequence homology to the long terminal repeats of three retroviruses, to two transposons and to the Alu family. This raises the possibility that the BCR repeat is involved in the t(9;22) as well as in generation of the BCR-related loci. Possible involvement of the p53 gene in clinical transition to acute phase was studied. In six patients and cell lines where one allele of the gene was deleted, the other allele was inactivated by loss of transcription, point mutation or rearrangement. The majority of patients, however, have both p53 alleles; detailed analysis of the p53 gene in several of them indicated normal transcription and amino acid sequence.

Delayed hypercalcemia with acute renal failure associated with nontraumatic rhabdomyolysis.

Delayed hypercalcemia developed in two patients with acute renal failure and rhabdomyolysis. In patient 1, the hypercalcemia appeared 14 days after the beginning of the diuretic phase of the illness and was associated with severe diastolic hypertension and soft-tissue calcification, including the lungs and skeletal muscles. The blood levels of 25-hydroxyvitamin D (25OHD3) were elevated during the hypercalcemia and decreased to normal when the patient became normocalcemic. In patient 2, the hypercalcemia occurred 55 days after the start of the diuretic phase and at a time when renal function was normal.

Serum thyroid hormone indexes in patients with primary hyperparathyroidism.

Serum total reverse triiodothyronine (rT3) levels are normal in patients with renal diseases with and without renal insufficiency but elevated in nonrenal nonthyroidal illnesses. To evaluate the role of secondary hyperparathyroidism of renal diseases in this difference, serum thyroid hormone levels were studied in 27 patients with primary hyperparathyroidism (PHP) and normal renal function. In PHP, total T3 levels were reduced (118 +/- 6 ng/dL, normal: 147 +/- 3 ng/dL) and correlated with PTH levels. Serum rT3 levels were also decreased (27 +/- 3 ng/dL, normal: 34 +/- 2 ng/dL). Values for serum total thyroxine (T4), T3 uptake ratio, free T4 index, and thyrotrophin were not altered. Serum rT3 levels were increased (63 +/- 13 ng/dL) in patients with hypercalcemia due to malignant neoplasms who had low T3 levels, undetectable PTH and normal renal function. Thus, PTH excess may be the factor responsible for the failure of rT3 levels to increase in PHP and secondary hyperparathyroidism.

REDD2-mediated inhibition of mTOR promotes dendrite retraction induced by axonal injury.

Dendritic defects occur in neurodegenerative diseases accompanied by axonopathy, yet the mechanisms that regulate these pathologic changes are poorly understood. Using Thy1-YFPH mice subjected to optic nerve axotomy, we demonstrate early retraction of retinal ganglion cell (RGC) dendrites and selective loss of mammalian target of rapamycin (mTOR) activity, which precede soma loss. Axonal injury triggered rapid upregulation of the stress-induced protein REDD2 (regulated in development and DNA damage response 2), a potent inhibitor of mTOR. Short interfering RNA-mediated REDD2 knockdown restored mTOR activity and rescued dendritic length, area and branch complexity in a rapamycin-dependent manner. Whole-cell recordings demonstrated that REDD2 depletion leading to mTOR activation in RGCs restored their light response properties. Lastly, we show that REDD2-dependent mTOR activity extended RGC survival following axonal damage. These results indicate that injury-induced stress leads to REDD2 upregulation, mTOR inhibition and dendrite pathology causing neuronal dysfunction and subsequent cell death.

Clinical and metabolic responses to parenteral nutrition in acute renal failure. A controlled double-blind study.

1. Thirty patients with acute renal failure who were unable to eat adequately were evaluated while they received parenteral nutrition with glucose alone (n = 7), glucose and 21 g/day essential amino acids (EAA, n = 11) or glucose, 21 g/day essential and 21 g/day nonessential amino acids (ENAA, n = 12). Energy intake did not differ with the three treatments. Patients were studied in a prospective double blind fashion. 2. Thirteen patients recovered renal function and 11 survived to leave the hospital. Those in whom renal failure was attributed to hypotension and/or sepsis had a poorer recovery of renal function (17%) and survival (17%). Recovery of renal function and survival was greater in patients on the medical service as compared to the surgical service and in those who received more energy. Recovery of renal function was worse in those treated with dialysis. There were no differences in recovery of renal function of survival among the three treatment groups. 3. Many patients were markedly catabolic as indicated by nitrogen balances, urea in nitrogen appearance rates (UNA), serum protein concentrations, and plasma amino acid levels. There was no correlation between the degree of catabolism and recovery of renal function or survival. Mean UNA in individual patients also correlated with body weight. Among the three groups, however, UNA was significantly less with the group receiving EAA as compared to ENAA. 4. Serum protein concentrations were lower than normal in all treatment groups. Serum albumin fell significantly during the treatment in the more catabolic patients. Plasma amino acid levels tended to fall in all three groups and concentrations at the end of the treatment were frequently lower than normal. 5. These data suggest that acute renal failure patients who are unable to eat adequately are often hypercatabolic and have a high mortality, particularly if hypotension or sepsis is the cause of renal failure. The improved survival in those with higher energy intakes, the high rate of net protein breakdown, the low serum protein levels and the reduced plasma concentrations of both essential and nonessential amino acids suggest that greater quantities of energy and both essential and nonessential amino acids may be beneficial to such patients.

Charcoal sorbent-induced hypolipidemia in uremia and diabetes.

Rats with hyperlipidemia associated with streptozotocin-induced diabetes or azotemia after subtotal nephrectomy were administered a diet containing 5% activated charcoal. Significant lowering of nonfasting serum cholesterol and triglyceride levels resulted. Charcoal-feeding also altered the abnormal high density lipoprotein electrophoresis pattern of diabetic rats toward normal.

Total parenteral nutrition in the management of acute renal failure.

Malnutrition is frequently present in patients with acute renal failure and may affect morbidity and mortality in this condition. When adequate nourishment cannot be given through the gastrointestinal tract, total parental nutrition with amino acids and hypertonic glucose may have beneficial results. Total parenteral nutrition has been reported to stabilize or reduce serum urea nitrogen, potassium and phosphorus levels, improve wound healing, enhance survival from acute renal failure, and possibly increase the rate of recovery of renal function. The optimal composition of the total parenteral nutrition infusate is unknown. Preliminary results of a double-blind study are reported in which one man received hypertonic glucose alone, two received glucose with essential amino acids (21 g/day), and three received glucose with essential (21 g/day) and nonessential (21 g/day) amino acids. All infusates were isocaloric. No differences were observed in serum urea nitrogen levels, serum urea nitrogen/creatinine ratios or urea appearance rates. Nitrogen balance was negative in all patients. The ratio of essential amino acids/nonessential amino acids were higher and the tyrosine/phenylalanine ratios were lower in plasma in the two patients receiving glucose with essential amino acids. No patient survived the hospitalization. In view of the markedly negative nitrogen balance frequently observed in these and earlier studies, the use of a different composition or quantity of amino acids, a higher energy intake, and anabolic hormones deserve further investigation.

Hyponatremia in hospitalized patients with the acquired immunodeficiency syndrome (AIDS) and the AIDS-related complex.

STUDY OBJECTIVE: To determine the frequency, etiology, and clinical association of hyponatremia in patients with the acquired immunodeficiency syndrome (AIDS) and AIDS-related complex (ARC). PATIENTS AND METHODS: A prospective analysis of 167 patients with AIDS and 45 patients with ARC admitted on 259 occasions to a large metropolitan teaching hospital during a 3-month period. RESULTS: Eighty-three patients (39%) with hyponatremia (serum sodium concentration less than 135 mmol/L) were observed during 99 hospitalizations, for a frequency of 38%. The mean (+/- standard error) of the lowest serum sodium concentration was 128 +/- 1 mmol/L in the hyponatremic patients and 138 +/- 1 mmol/L in the normonatremic patients. Hyponatremia was present on admission during 57 hospitalizations and was associated with gastrointestinal losses and hypovolemia in 43%. When hyponatremia developed during hospitalization, 68% of the patients were clinically euvolemic and had a syndrome consistent with inappropriate secretion of antidiuretic hormone (SIADH). Patients with hyponatremia were hospitalized longer than those with normal serum sodium concentrations (17 +/- 1 versus 9 +/- 1 days, p < 0.001). In addition, the mortality rate in the hyponatremic group was higher than that in the normonatremic group (36.5% versus 19.7%, p < 0.01). CONCLUSION: Hyponatremia is a common electrolyte disorder in patients hospitalized with AIDS or ARC and is frequently associated with gastrointestinal losses or SIADH as well as increased morbidity and mortality.

Acquired cystic disease of the kidneys. Computed tomography and ultrasonography appraisal in patients on peritoneal and hemodialysis.

Screening chronic hemodialysis patients (CHD) for acquired cystic disease of the kidneys (ACDK) and its complications (hemorrhage and neoplasm) has become accepted management. We evaluated patients on CHD as well as patients on chronic peritoneal dialysis (CPD) for ACDK. The kidneys of 80 chronic dialysis patients were examined by CT and real time sonography. Forty-four were hemodialysis and 36 were peritoneal dialysis patients. ACDK was found in more than 90% of both CHD and CPD patients who had been dialyzed longer than three years. Bilateral renal carcinoma was detected in one hemodialysis patient. Our results show that chronic peritoneal dialysis patients are also at risk for ACDK and its associated complications. A similar natural history for the development of ACDK in both forms of dialysis suggests that the same screening precautions should be instituted for chronic peritoneal dialysis patients.

Total parenteral nutrition with high or low nitrogen intakes in patients with acute renal failure.

This study was undertaken to assess the clinical and metabolic responses to total parenteral nutrition (TPN) in patients with acute renal failure who could not be nourished adequately through the enteral tract. The TPN provided either about 21 g/day of essential amino acids (EAA) or a larger quantity of essential and nonessential amino acids (ENAA); the ratio of essential to nonessential amino acids in the latter preparation was 1.0:1.0. Attempts were made to give sufficient ENAA nitrogen to equal or slightly exceed the urea nitrogen appearance (UNA). Five patients were randomly assigned to receive TPN with EAA (2.3 g of nitrogen per day) and six patients to receive ENAA (11.3 g of nitrogen per day). Hypotension with trauma or infarcted intestine was the cause of acute renal failure in 10 of the 11 patients. Three of the five patients receiving EAA recovered renal function, and two survived. In the patients receiving ENAA, as compared with those given EAA, UNA was significantly greater (14 +/- 7.4 [SD] vs. 7.5 +/- 3.0 g/day; P less than 0.01), and nitrogen balance, estimated from the difference between intake nitrogen and UNA was slightly, but not significantly, less negative (-3.0 +/- 4.0 vs. -5.2 +/- 2.9 g of nitrogen per day). These preliminary findings suggest that in comparison to TPN with EAA, there is no advantage to larger amounts of ENAA (76 +/- 13 g/day). Studies are indicated to assess whether a multifaced approach using TPN with ENAA and possibly a larger proportion of the branched-chain amino acids, higher energy intakes, anabolic agents, and continuous arterio-venous hemofiltration will improve morbidity and mortality in patients with acute renal failure.

Charcoal-induced lipid reduction in uremia.

Charcoal, in divided oral doses totalling 35 g/day, was administered to six patients with renal insufficiency (creatinine clearance of 0 to 45 ml/min). Significant (P less than 0.01) reductions in serum cholesterol and triglycerides were observed in the three most hyperlipidemic patients. Maximal decreases in charcoal responders, as compared with control values, were for cholesterol (43%, 23.4% and 40.4%) and for triglycerides (76%, 60.3% and 64.3%). None of the patients showed altered concentrations of BUN, serum creatinine, uric acid, or vitamin A. Because of its safety and the profundity of its hypolipidemic action, it is suggested that charcoal may find applicability in the management of azotemic diabetic and nephrotic hyperlipidemia.

Alterations of serum reverse triiodothyronine and thyroxine kinetics in chronic renal failure: role of nutritional status, chronic illness, uremia, and hemodialysis.

atients with end-stage chronic renal failure (CRF) and those receiving dialysis therapy have normal or decreased serum total T4 (TT4), reduced serum total T3 (TT3), and normal total reverse T3 (TrT3) levels. Those with nonrenal nonthyroidal illnesses or malnutrition have low TT4 and TT3 but elevated TrT3 values. To evaluate the mechanism(s) for the normal TrT3 levels in CRF, we performed intravenous bolus kinetic studies of rT3 and T4 in patients with CRF, in those treated with chronic hemodialysis, in patients with nonrenal nonthyroidal illnesses, and in normal subjects. The CRF patients were selected to have good nutritional status as indicated by normal serum transferrin, relative body weight, and body mass index values. The CRF patients had normal TrT3, TT4, and free T4 values, increased free fraction of rT3, free rT3, and thyroxine-binding globulin levels, and decreased TT3 concentrations. Noncompartmental analysis of the rT3 kinetics indicated normal production rate, reduced cellular clearance rate, and increased pool size and residence time values in both the CRF and nonrenal patients. In CRF, the serum clearance rate was normal, but the fractional rate of exit, permeability, extravascular binding, and the apparent volume of distribution were increased. In contrast, the nonrenal patients had reduced serum clearance rate, permeability, and extravascular binding, whereas the fractional rate of exit and apparent volume of distribution were not significantly altered. The T4 kinetics in CRF paralleled those of the nonrenal patients, with a reduced fractional rate of exit and permeability in both groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Deliverability of psychoeducational family management.

As part of an open clinical trial currently underway at the Max Planck Institute of Psychiatry in Munich, the feasibility of behavioral family management (Falloon et al. 1984) for schizophrenia in combination with two different neuroleptic medication strategies was investigated. The treatment approaches were psychoeducational family management with a standard dose or with targeted medication. In this article the following questions were addressed: (1) What proportion of the total schizophrenia population admitted as inpatients might be eligible for psychoeducational family treatment (assessment based on n = 411 over a 33-month period)? (2) How representative of this population are the patients who were randomized to the experimental groups? (3) How many patients dropped out of treatment after entering the trial? The results show that about 60 percent (247) of the patients were eligible for a psychoeducational treatment approach. Of these, 34 percent (85) participated in the trial and were randomized to the treatments. Only 4 percent of the relatives but 20 percent of the patients refused to take part in the study. The 85 trial patients did not differ from the total eligible on the numerous socioeconomic and symptom variables assessed. The treatment dropout rate was 11 percent. Those patients who accepted treatment did not differ from those patients who dropped out on socioeconomic or illness variables. The results indicate that early identification of dropouts is not possible at least with the methods used in this study.

Cross-national reliability, concurrent validity, and stability of a brief method for assessing expressed emotion.

The Five-Minute Speech Sample (FMSS; Magana et al., 1986) is a brief method designed to assess the "expressed emotion" (EE) status of a respondent. The FMSS-EE rating is derived from statements made by a patient's key relative during a 5-minute monologue, with codes similar to those used in the original EE rating system (e.g., criticism and emotional overinvolvement). Rating is done from the audiotape and takes about 15-30 minutes. The article reports on the results of a cross-national study carried out in Germany using the FMSS with a sample of 60 relatives of schizophrenic patients. Results indicated that (1) German investigators could readily learn the system and could achieve a high degree of interrater reliability; (2) the association with the original index of EE, the Camberwell Family Interview, was comparable to that found by Magana et al.; and (3) the method yields very stable data over a 4-5 week retest period.