Alternative forms of Max as enhancers or suppressors of Myc-ras cotransformation.

Max is a basic-helix-loop-helix-leucine zipper protein capable of forming sequence-specific DNA binding complexes with Myc proteins. An alternatively spliced messenger RNA has been identified that encodes a form of Max truncated at the COOH-terminus. This delta Max protein retained the ability to bind to the CACGTG motif in a complex with c-Myc but lacks the nuclear localization signal and the putative regulatory domain of Max. When tested in a myc-ras cotransformation assay in rat embryo fibroblasts, Max suppressed, whereas delta Max enhanced, transformation. Thus, the max gene may encode both a negative and a positive regulator of c-Myc function.

Determination of sequences responsible for the differential regulation of Myc function by delta Max and Max.

The DNA-binding, transcriptional activation and transforming activities of the Myc protein require dimerization with Max. Max can form also homodimers which are able to bind the same DNA sequence as Myc/Max heterodimers and suppress Myc-induced transcription and transformation. We have recently identified a naturally occurring truncated form of Max, delta Max, which in a rat embryo fibroblast enhances transformation by Myc and Ras. Like Max, this delta Max protein contains a b-HLH-Zip domain, except that the end of the leucine zipper is replaced by five delta Max-specific amino acid residues. Delta Max also lacks the C-terminal sequences of Max including a nuclear localisation signal. Here we have dissected the regions responsible for the specific effects of Max and delta Max in Ras-Myc cotransformation of rat embryo fibroblasts. Our results indicate that the suppressive activity of Max requires C-terminal acidic and basic regions and an intact leucine zipper. Replacement of the end of the leucine zipper with the delta Max-specific sequence is responsible for the enhancement of transformation by delta Max. Surprisingly, delta Max does not require the DNA-binding basic region for enhancement of transformation and has no effect on Myc-induced transcription activation from Myc/Max-binding site-containing promoter construct.

The PIM-1 serine kinase prolongs survival and inhibits apoptosis-related mitochondrial dysfunction in part through a bcl-2-dependent pathway.

We have examined potential mechanisms by which the Pim-1 kinase acts as a hematopoietic cell survival factor. Enforced expression of the wild type 33 kd (FD/hpim33) and 44 kd (FD/mpim44) Pim-1 proteins in murine factor-dependent FDCP1 cells prolonged survival after withdrawal of IL-3, while expression of a dominant negative Pim-1 protein (FD/pimNT81) shortened survival. Following removal of IL-3 FDCP1 cells exhibited loss of mitochondrial transmembrane potential and production of reactive oxygen species, as determined by flow cytometry analysis. The wild type Pim-1 proteins decreased these changes while the dominant negative protein enhanced mitochondrial dysfunction. The antiapoptotic activity of the kinases could not be attributed to modulation of glutathione, catalase, or superoxide dismutase activities. Both the FD/hpim33 and FD/mpim44 cells maintained expression of bcl-2 mRNA following cytokine removal, while a substantial decrease was seen in FD/neo cells. To modulate Bcl-2 protein levels, a bcl-2 antisense RNA construct was coexpressed with the wild type pim-1 cDNAs. FD/hpim33 cells with low cellular Bcl-2 protein levels had shortened cytokine-independent survival compared with FD/hpim33 clones with high Bcl-2 expression. However survival of FD/mpim44 cells after IL-3 withdrawal was substantially independent of cellular Bcl-2 protein levels. The 33 kd protein delayed, and the 44 kd protein completely prevented enhanced cell death associated with enforced expression of human Bax protein however. Our results suggest that the 33 kd Pim-1 kinase may enhance cell survival through cooperation with and regulation of bcl-2. In addition the 44 kd kinase may regulate the expression or activity of other pro- and anti-apoptotic members of the bcl-2 family.

Alternative mRNA forms and open reading frames of the max gene.

The max gene encodes a heterodimeric partner of Myc. We have recently identified an alternative max mRNA (delta max) that contains an additional internal exon introducing an in-frame translational termination. Here we have studied the expression of human max mRNAs by Northern blotting analysis. In addition to the major 2.3-kb mRNA form, four bands were identified. Our results indicate that these bands represent differentially spliced mRNA forms, which contain altogether three open reading frames. In addition to the previously identified Max and delta Max proteins, sequence analysis of a 3.5-kb mRNA form predicted a protein that resembles delta Max in structure. Like delta Max, this protein enhanced the number of transformed foci in the ras-myc co-transformation assay. Although the 3.5-kb mRNA represents a minor form in actively proliferating cells, a shift from the major 2.3-kb mRNA to the 3.5-kb form was observed in response to high cell density or acidification of the growth medium. Our results indicate the presence of several differentially spliced mRNA forms of the max gene, and suggest a possible mechanism for the production of functionally distinct Max proteins.

Developmental expression of pim kinases suggests functions also outside of the hematopoietic system.

We have cloned a novel quail cDNA with strong homology to the pim family of proto-oncogenes. The deduced amino acid (aa) sequence of the cDNA, named qpim, is more closely related to Xenopus Pim and to the recently identified rat Pim-3 than to human or rodent Pim-1 or Pim-2. The protein encoded by the qpim cDNA can autophosphorylate itself and share substrates with murine Pim-1, suggesting functional redundancy to other Pim family serine/threonine kinases. We have compared the expression of qpim in avian embryos to mouse pim-1, -2 and -3 by in situ hybridization. qpim shows a highly dynamic expression pattern, particularly at early developmental stages. Surprisingly, its expression pattern is not identical to any of the murine pim genes, which show complementary and/or partially overlapping expression sites both in- and outside of the hematopoietic system. Altogether, our results suggest novel functions for Pim family kinases during embryonic development, in particular in epithelia and in the central nervous system.

Downregulation of the early genomic growth factor response in neu oncogene-transformed cells.

Peptide growth factor-induced signal transduction leads to a long-term adjustment of the genetic programs of responding cells. A point mutation in the transmembrane domain of the neu receptor has been found to activate its tyrosine kinase and oncogenic potential. Our previous studies show that ligand stimulation of a chimeric epidermal growth factor receptor-neu proto-oncogene (EGF-R/neu) induces the neu tyrosine kinase and leads to the programmed activation of cell growth-regulated genes. We have now studied the effect of the neu oncoprotein on the genomic growth factor response in cells expressing the EGF-regulated neu tyrosine kinase. Expression of the neu oncogene in these cells inhibited 75-90% of the EGF-stimulated mRNA induction of the immediate early serum response genes, such as junB encoding a transcription factor, N10 encoding a putative nuclear hormone binding receptor for an as yet undefined ligand, and B10, the protein product of which is still unknown. The relative lack of mRNA induction was not due to a loss of the chimeric EGF-R/neu receptors from the cell surface. Also, the neu oncogene decreased serum- and tumor promoter induction of these genes. Our results suggest that the neu oncogene is capable of deregulating mRNA responses to extracellular signalling, similar to the effects of the c-Ha-ras oncogene. Knowledge of the mechanisms responsible for these changes in gene regulation will help to define oncogenic transformation of cells in molecular terms.

Coronary flow reserve is reduced in young men with IDDM.

Disturbances of coronary circulation have been reported in diabetic patients with microvascular complications but without obstructive coronary atherosclerosis. The aim of the present study was to investigate coronary flow reserve in young adult patients with IDDM but without microalbuminuria and diabetic autonomic neuropathy. Coronary flow reserve was determined in 12 nonsmoking male patients with IDDM (age 30.0 +/- 6.6 years) and 12 healthy matched volunteers. Groups were similar with respect to blood pressure and serum lipid concentrations, and no subject had a positive family history of coronary heart disease. The patients with IDDM had normal exercise echocardiography and autonomic nervous function tests. Five patients had minimal background retinopathy, and none had microalbuminuria. Positron emission tomography and [15O]H2O were used to measure myocardial blood flow at rest and after dipyridamole administration. The studies were performed during euglycemic hyperinsulinemia (serum insulin approximately 70 mU/l). The baseline myocardial blood flow was similar in patients with IDDM and in control subjects (0.84 +/- 0.18 vs. 0.88 +/- 0.25 ml x g(-1) x min(-1), NS). The myocardial blood flow during hyperemia was 29% lower in patients with IDDM (3.17 +/- 1.57) compared with the control subjects (4.45 +/- 1.37 ml x g(-1) x min(-1), P < 0.05). Consequently, coronary flow reserve (the ratio of flow during hyperemia and at rest) was lower in diabetic patients than in control subjects (3.76 +/- 1.69 vs. 5.31 +/- 1.86, P < 0.05) and the total coronary resistance during hyperemia was higher in diabetic patients (53.7 +/- 31.5) compared with the control subjects (31.4 +/- 11.6 mmHg x min x g x ml(-1), P < 0.05). The coronary flow reserve was similar in diabetic patients with and without mild background retinopathy. No association was found between the coronary flow reserve and serum lipid or HbA1c values in either group. Coronary flow reserve is impaired in young adult males with IDDM and no or minimal microvascular complications and without any evidence of coronary heart disease. This abnormality cannot be explained by standard coronary heart disease risk factors. The results imply early impairment of coronary vascular reactivity in IDDM patients, which may represent an early precursor of future coronary heart disease or may contribute to the pathogenesis of diabetic cardiomyopathy.

Glucagon-stimulated and postprandial plasma C-peptide values as measures of insulin secretory capacity.

Basal, postprandial (2 h after breakfast), and glucagon-stimulated plasma C-peptide concentrations were determined in a group of 36 adult diabetic patients. Basal and postprandial C-peptide values were measured on consecutive days to estimate the degree of variation of C-peptide secretion. In a subgroup of 15 diabetic patients treated chronically with diet and oral hypoglycemic agents (sulfonylureas or a combination of sulfonylureas and metformin), we studied whether administration of sulfonylureas immediately before breakfast had any effect on postprandial C-peptide values. Absolute differences between two consecutive fasting C-peptide concentrations in insulin-requiring patients were less than 0.1 nM in all but 1 patient, in whom the difference was 0.18 nM. In subjects treated with oral hypoglycemic agents the median difference was 0.12 nM (range 0-0.38 nM). Absolute differences between two consecutive postprandial C-peptide concentrations were all less than 0.1 nM in insulin-requiring patients. No significant difference was found between postprandial C-peptide concentrations with or without preceding administration of oral hypoglycemic agents (medians 1.35 and 1.30 nM, respectively). Glucagon-stimulated C-peptide concentrations were somewhat higher than the postprandial values. However, equal discrimination between insulin-requiring and non-insulin-requiring diabetic patients was found by measuring postprandial or glucagon-stimulated C-peptide concentrations.

Elevated serum total renin is insensitive in detecting incipient diabetic nephropathy.

OBJECTIVE: To assess the influence of incipient diabetic nephropathy on the levels of total renin in serum. RESEARCH DESIGN AND METHODS: Fifty-five adult patients with insulin-dependent diabetes mellitus (IDDM) were examined in a cross-sectional study. The main outcome measures were serum total renin concentration and urinary albumin excretion rate. RESULTS: The total renin concentrations in serum were significantly (P < 0.05) higher in 12 patients with microalbuminuria than in 43 patients without albuminuria, but this difference was significant only in men. There was a significant but weak positive correlation between urinary albumin excretion rate and serum total renin concentration in all patients (r = 0.412, P < 0.05, n = 55), but the sensitivity of high serum concentrations of total renin in detecting incipient nephropathy was low (17%). In the study group, two of the three patients suffering from proliferative retinopathy had the highest total renin concentrations in serum. CONCLUSIONS: Microalbuminuric patients have higher mean serum total renin concentrations than normoalbuminuric patients, but because of low sensitivity, high total renin concentration cannot be used for screening incipient diabetic nephropathy.

Comparison of encainide and quinidine for supraventricular tachyarrhythmias.

The antiarrhythmic efficacy and tolerability of encainide and quinidine were compared in an open-design study in which 50 patients with recurrent supraventricular tachyarrhythmias received intravenous encainide in an initial phase and oral encainide and quinidine in a subsequent, randomized crossover phase. Oral encainide (75 to 200 mg/day), administered to 47 patients for an average of 4.7 months, was effective in 77%, and oral quinidine (1,200 mg/day), administered to 44 patients for an average of 3.2 months, was effective in 66% of the patients (difference not significant). When the duration of therapy at each crossover period was compared, the percentage of patients who continued to take encainide was consistently higher than the percentage who continued to take quinidine (p less than 0.01). Twenty-nine percent of the patients discontinued encainide treatment, 23% because of clinical inefficacy and 6% because of adverse effects. Fifty-nine percent of the patients discontinued quinidine treatment, 20% because of inefficacy and 39% because of adverse effects. Based on antiarrhythmic efficacy, encainide is at least as effective as quinidine in the treatment of patients with supraventricular tachyarrhythmias. However, because of encainide's much greater tolerability, it was distinctly superior in terms of clinical use. This study was an open-design, randomized crossover trial to compare the efficacy and tolerability of encainide with those of quinidine in the treatment of supraventricular tachyarrhythmias.

Myc amplification: regulation of myc function.

The myc oncogenes have been implicated in the control of cell proliferation in both normal and neoplastic cells. There is increasing evidence that Myc proteins function as transcriptional regulators of other genes apparently involved in the control of cell proliferation. The effects of Myc on both gene expression and cell growth are differentially regulated by the recently described Max and delta Max proteins that can either cooperate or compete with Myc for sequence-specific DNA binding.

Diabetic complications are associated with liver enzyme activities in people with type 1 diabetes.

This study was performed to clarify if diabetic complications are associated with liver enzyme activities in type 1 diabetic outpatients. Elevated activities of serum aminotransferases are a common sign of liver disease and are observed more frequently among people with diabetes than in the general population. Many studies have shown an association between specific diabetic complications and disturbances in various tissues, such as diabetic nephropathy and cardiovascular diseases, but only limited data are available on the possible association between diabetic complications and liver function. We studied 28 patients with type 1 diabetes. Mean age was 43.4+/-9.5 (S.D.), and duration of diabetes 25.2+/-9.7. Limited joint mobility (LJM) was assessed by the Rosenbloom's method. Background and proliferative retinopathy, and peripheral symmetrical polyneuropathy were also assessed. Activities of alanine amino transferase (ALT), gamma-glutamyl transferase (GGT) and alkaline phosphatase (ALP) in serum were determined. The metabolic control of the diabetes was evaluated by the glycosylated haemoglobin A(1c) (HbA(1c)) level and lipid values were also measured. ALT activity was associated with LJM (P<0.01) and with neuropathy (P<0.01). Association between GGT activity and LJM (P<0.01) and neuropathy (P<0.01) were also found. GGT activity was also associated with the severity of retinopathy (P<0.01). None of these associations was explained by confounding effects of diabetes duration, age, body mass index (BMI), HbA(1c) or alcohol consumption. In conclusion, diabetic complications such as LJM, retinopathy and neuropathy are associated with liver enzyme activities independent of alcohol consumption, BMI and metabolic control of diabetes.

Dupuytren's disease in type I diabetic subjects: investigation of biochemical markers of type III and I collagen.

OBJECTIVE: To clarify whether biochemical markers of collagen type III and I metabolism show alterations in type I diabetic subjects with Dupuytren's disease (DD) compared to those without DD. METHODS: DD was assessed in a total of 28 type I diabetic subjects, mean age 43.4 +/- 9.5 (SD) and duration of diabetes 25.2 +/- 9.7 years. Concentrations of aminoterminal propeptide of type III procollagen (PIIINP), carboxyterminal propeptide of type I procollagen (PICP) and carboxyterminal cross-linked telopeptide of type I collagen (ICTP) in serum and excretion of cross-linked N-telopeptides of type I collagen (NTX) and deoxypyridinoline crosslinks (DPyr) into urine were measured. RESULTS: The prevalence of DD was 32% (9 of 28 diabetic subjects). Average serum ICTP was 2.7 +/- 0.8 micrograms/l in subjects without DD and 3.6 +/- 1.2 micrograms/l with DD (p = 0.0276). No significant association between other collagen markers and DD was found. The reference intervals of PIIINP and ICTP were exceeded only in 1 and 2 subjects, respectively, and they both had DD. CONCLUSION: The degradation of type I collagen might be increased in diabetic subjects with DD. The overall implication was that synthesis or degradation of type III and I collagen in diabetic subjects with DD did not differ enough from those without DD to reflect changes in the biochemical markers of type III and I collagen.

Biochemical markers of types I and III collagen and limited joint mobility in type 1 diabetic patients.

Limited joint mobility (LJM), a long-term complication of diabetes, has been shown to be associated with microvascular complications of diabetes. Connective tissue alterations may contribute to the development of LJM and other diabetic complications. We tested whether biochemical markers of types I and III collagen metabolism are associated with LJM in type 1 diabetes. We studied 28 male patients of mean age 43.4 years (SD=9.5) and with a duration of diabetes of 25.2 years (SD=9.7) years. LJM assessment included goniometric measurements of the joints and classification by Rosenbloom's method. We measured serum concentrations of aminoterminal propeptide of type III procollagen (PIIINP), carboxyterminal propeptide of type I procollagen (PICP) and carboxyterminal crosslinked telopeptide of type I collagen (ICTP); urinary excretion of crosslinked N-telopeptides of type I collagen (NTX) and deoxypyridinoline crosslinks (DPyr) was also measured. Although average serum PIIINP tended to be higher in subjects with moderate-severe LJM (3.1 +/- 1.3 microg/l) than in subjects with mild LJM (2.5 +/- 0.7 microg/l) or without LJM (2.6 +/- 0.4 microg/l), no significant association was found (p<0.27). Concentrations of the other collagen markers were not different in subjects with or without LJM. We conclude that synthesis and degradation of types I and III collagen in diabetic subjects with LJM did not differ from those without LJM to reflect changes in the biochemical markers of these proteins.

Immunohistochemical labelling for prostate specific antigen in non-prostatic tissues.

Immunohistochemical detection of prostate specific antigen (PSA) in metastases of adenocarcinomas is widely used as an aid to identify the prostatic origin of metastatic cells. However, on the one hand, PSA may not be expressed in some poorly differentiated prostatic carcinomas, while on the other, PSA immunoreactivity has been found in small amounts in non-prostatic tissues. The aim of the current study was to evaluate the prevalence of PSA immunoreactivity in normal non-prostatic tissues and in breast carcinoma. PSA was localized by immunohistochemistry with four commercial antibodies in 34 different normal human tissues, and in 15 ductal and seven apocrine breast carcinomas. Concentrations of PSA in tissue homogenates of prostate and nine non-prostatic tissues from autopsied subjects were measured by a two-site immunoradiometric assay. Weak PSA immunoreactivity was found by immunohistochemistry in kidney, parotid gland and pancreatic tissues. Variable PSA immunoreactivity was seen in three cases of ductal (20%) and two cases of apocrine breast carcinoma (28%). No consistent PSA immunoreactivity was found in homogenates of non-prostatic tissues by the immunoradiometric assay. We conclude that PSA is a quite specific marker of prostatic tissue. However, there are some non-prostatic neoplastic and normal tissues that express PSA. Therefore, a definite diagnosis of metastasis of prostatic origin cannot be made on the basis of immunolabelling for PSA alone.

Importance of storing emergency serum samples for uncovering murder with insulin.

A case of a previously healthy 48-year-old man murdered by exogenous insulin administration is reported. The patient was delivered unconscious to the emergency unit. Initially, treatment with hyperbaric oxygen was commenced because decompression sickness was suspected. However, the treatment was aborted as the patient was found to be hypoglycaemic (nadir serum glucose 0.3 mmol/l) and treatment and diagnostics of hypoglycaemia commenced. Brain damage due to hypoglycaemia was severe, and the patient remained in a vegetative state for 2 months before he died of multiorgan failure. Serum samples drawn at admittance were stored frozen, whereby it was possible to show retrospectively, that while the concentration of insulin in serum was high (75 mU/l, increasing further to over 240 mU/l in the next few hours) concentration of C-peptide was low (below detection limit of 0.1 nmol/l) at the hypoglycaemic stage. It was concluded that the patient had received exogenous insulin somehow, and the police was informed. Circumstantial evidence obtained during ensuing criminal investigation was considered by the court to prove the patient's wife (a nurse) guilty of murder. The availability of stored frozen serum samples drawn at the early stage of hospitalization helped to uncover the crime involved in our case.

Kaposi's sarcoma herpesvirus lytic replication compromises apoptotic response to p53 reactivation in virus-induced lymphomas.

Primary effusion lymphomas (PELs) are aggressive Kaposi's sarcoma herpesvirus (KSHV)-induced malignancies with median survival time <6 months post-diagnosis. Mutations in the TP53 gene seldom occur in PELs, suggesting that genetic alterations in the TP53 are not selected during PEL progression. We have reported that p53 reactivation by an inhibitor of the p53-MDM2 interaction, Nutlin-3, induces selective and massive apoptosis in PEL cells leading to efficient anti-tumor activity in a subcutaneous xenograft model for PEL. Here, we show compelling anti-tumor activity of Nutlin-3 in the majority of intraperitoneal PEL xenografts in vivo. Interestingly, our results demonstrate that spontaneous induction of viral lytic replication in tumors could drastically attenuate the p53-dependent apoptotic response to Nutlin-3. Moreover, viral reactivation compromised p53-dependent apoptosis in PEL cells treated with genotoxic anti-cancer agents doxorubicin and etoposide. We have recently demonstrated that the Ser/Thr kinases Pim 1 and 3 are required to trigger induction of the lytic replication cascade of KSHV. We have now assessed the ability of a novel Pim kinase inhibitor to restore the Nutlin-3-induced cytotoxicity in lytic PEL cells. PEL cells induced to lytic replication by phorbol esters showed 50% inhibition of active viral replication following treatment with the Pim kinase inhibitor. Importantly, co-treatment of these cells with the kinase inhibitor and Nutlin-3 resulted in a robust restoration of the Nutlin-3-induced cell death. These results highlight the potential impact of activation of viral lytic replication on disease progression and response to treatment in KSHV-induced lymphomas.

Role of myc amplification and overexpression in cell growth, differentiation and death.

Genes of the myc family are apparently the most intensively studied of all nuclear oncogenes. This is because their expression is deregulated in many types of human neoplasia due to chromosomal translocation or gene amplification, and because their exact roles in the regulation of cell proliferation have remained poorly known. However, the recent characterization of several domains in Myc proteins that enable sequence-specific regulation of other growth-related genes, and the identification of proteins interacting with Myc proteins have provided insight into the function(s) of Myc proteins in both normal and neoplastic cells. While the natural target genes for Myc remain to be identified, it has become evident that myc overexpression not only promotes cell proliferation, but also increases the rate of programmed cell death.

Immediate early gene responses of NIH 3T3 fibroblasts and NMuMG epithelial cells to TGF beta-1.

Transforming growth factor beta has a wide range of physiological effects on cell growth and metabolism. We have previously reported on the rapid induction of jun transcription factors in TGF beta-treated cells. Here we show that the early genomic response to TGF beta-1 includes activation of a broad spectrum of serum-inducible genes both in NIH 3T3 fibroblasts and in NMuMG epithelial cells, which are growth-stimulated and growth-inhibited by TGF beta, respectively. Of particular interest is the presence of a putative nuclear DNA-binding receptor (N10) and zinc finger transcription factors (Krox 20 and Krox 24) among the TGF beta-induced genes. In addition to the stimulatory effects of TGF beta, expression of a few genes including c-myc is decreased in both types of cells. In cells transformed by neu or ras oncogenes the immediate early mRNA responses to TGF beta are deregulated. Our results suggest that certain transcription factors are required for both positive and negative regulation of cell proliferation by TGF beta, and that their relative concentrations may determine the subsequent cellular responses.