Genetic dissection of c-myc apoptotic pathways.

All biological functions mediated by the c-myc oncoprotein require an intact transactivation domain (TAD). We compared TAD mutants for their ability to promote apoptosis of 32D myeloid cells in response to interleukin-3 (IL-3) deprivation and exposure to chemotherapeutic drugs, and to activate ornithine decarboxylase, an endogenous c-myc target. Different sub-regions of the TAD were required to mediate each function. cDNA microarrays were then used to identify multiple c-myc-regulated transcripts, some of which were also modulated by IL-3 or cytotoxic drugs, as well as by specific sub-regions of the TAD. Several of the c-myc-regulated transcripts had also been previously identified as targets for IFN-gamma. The functional consequences of their deregulation were manifested by a marked sensitivity of c-myc-overexpressing cells to IFN-gamma-mediated apoptosis. Our results establish that several well-characterized functions of c-myc are separable and correlate with the expression of a novel group of target genes, some of which also mediate the apoptotic action of IFN-gamma.

MYC oncogenes and human neoplastic disease.

c-myc, N-myc and L-myc are the three members of the myc oncoprotein family whose role in the pathogenesis of many human neoplastic diseases has received wide empirical support. In this review, we first summarize data, derived mainly from non-clinical studies, indicating that these oncoproteins actually serve quite different roles in vivo. This concept necessarily lies at the heart of the basis for the observation that the deregulated expression of each MYC gene is reproducibly associated with only certain naturally occurring malignancies in humans and that these genes are not interchangeable with respect to their aberrant functional consequences. We also review evidence implicating each of the above MYC genes in specific neoplastic diseases and have attempted to identify unresolved questions which deserve further basic or clinical investigation. We have made every attempt to review those diseases for which significant and confirmatory evidence, based on studies with primary tumor material, exists to implicate MYC members in their causation and/or progression.

Differential apoptotic behaviors of c-myc, N-myc, and L-myc oncoproteins.

c-, N-, and L-myc are related nuclear oncoproteins that bind similar DNA sites and cooperate with activated ras oncogenes to transform primary fibroblasts. Although c-myc can also promote apoptosis in some cells after growth factor withdrawal or exposure to cytotoxic agents, roles for N- and L-myc in apoptosis remain undetermined. To address this, c-, N-, or L-myc were stably expressed in the interleukin 3 (IL-3)-dependent 32D hematopoietic cell line. The apoptotic response of each cell line was assessed after IL-3 withdrawal or treatment with four structurally unrelated cytotoxic agents. All three oncoproteins accelerated apoptosis after IL-3 withdrawal. In contrast, whereas c-myc overexpression generally sensitized cells to cytotoxic drugs, N-myc and L-myc overexpression produced resistance. myc expression tended to be associated with a more robust G2-M arrest after drug exposure, but this did not correlate with drug sensitivity or resistance. Bcl-2 and Bcl-X(L) protected control cells against apoptosis after either IL-3 withdrawal or drug exposure, although in some cases this effect could be overridden by myc oncoproteins, particularly N-myc and L-myc. Our results suggest that the apoptotic pathways activated upon IL-3 withdrawal and cytotoxic drug treatment are distinct and differentially affected by members of the myc and Bcl-2 families.

Distinct apoptotic responses imparted by c-myc and max.

The c-myc oncoprotein accelerates programmed cell death (apoptosis) after growth factor deprivation or pharmacological insult in many cell lines. We have shown that max, the obligate c-myc heterodimeric partner protein, also promotes apoptosis after serum withdrawal in NIH3T3 fibroblasts or cytokine deprivation in interleukin-3 (IL-3)-dependent 32D murine myeloid cells. We now show that c-myc- and max-overexpressing 32D cells differ in the nature of their apoptotic responses after IL-3 removal or treatment with chemotherapeutic compounds. In the presence of IL-3, c-myc overexpression enhances the sensitivity of 32D cells to Etoposide (Sigma, St Louis, MO), Adriamycin (Pharmacia, Columbus, OH), and Camptothecin (Sigma), whereas max overexpression increases sensitivity only to Camptothecin. Drug treatment of c-myc-overexpressing cells in the absence of IL-3 did not alter the spectrum of drug sensitivity other than to additively accelerate cell death. In contrast, enhanced sensitivity to Adriamycin, Etoposide, and Taxol (Bristol-Meyers Squibb, Princeton, NJ) was revealed in max-overexpressing cells concurrently deprived of IL-3. Differential rates of apoptosis were not strictly correlated with the ability of the drugs to promote G1 or G2/M arrest. Ectopic expression of Bcl-2 or Bcl-XL blocked drug-induced apoptosis in both cell lines. In contrast, whereas Bcl-2 blocked apoptosis in both cell lines in response to IL-3 withdrawal, Bcl-XL blocked apoptosis in max-overexpressing cells but not in c-myc-overexpressing cells. These results provide mechanistic underpinnings for the idea that c-myc and max modulate distinct apoptotic pathways.

Diabetic foot infections. Bacteriology and activity of 10 oral antimicrobial agents against bacteria isolated from consecutive cases.

OBJECTIVE: To study the relative frequency of bacterial isolates cultured from community-acquired foot infections and assess their comparative in vitro susceptibility to sparfloxacin, levofloxacin, and eight other commonly used oral antimicrobial agents. RESEARCH DESIGN AND METHODS: This is a prospective study in which the infected wounds of 25 consecutive diabetic patients seen by one of the authors were cultured as they entered the hospital. Isolates were stored and tested for susceptibility to 10 oral antimicrobial agents using the agar dilution method. RESULTS: Staphylococcus aurcus was the most common isolate (76% of patients), including methicillin-resistant S. aurcus (MRSA) in 5 of 25 (20%) patient wounds. Streptococci, enterococci, Enterobacteriaceae, and anaerobes were also present in > or = 40% of patient wounds. Sparfloxacin and levofloxacin were the most active agents tested with activity against > or = 88% of isolates. Isolates resistant to sparfloxacin and levofloxacin included MRSA, enterococci, and some anaerobes. When analyzed by prior exposure to antibiotics, patients who had previously received oral antibiotics were more likely to have MRSA, enterococci, and Pseudomonas aeruginosa isolated and less likely to have Enterobacteriaceae and anaerobes isolated from their wounds. CONCLUSIONS: MRSA and enterococci are now a common cause of diabetic foot infections, and the increased prevalence may be due to antimicrobial use. These wounds may require use of combined antimicrobial therapy for initial outpatient management. The new fluoroquinolones, sparfloxacin and levofloxacin, were the most active oral agents tested.

Transcriptional regulation of repressor synthesis in mycobacteriophage L5.

Mycobacteriophage L5 is a temperate phage of the mycobacteria that forms stable lysogens in Mycobacterium smegmatis. Lysogeny is maintained by the putative repressor, the gene 71 product, which also mediates immunity to superinfection. We show here that there are three promoters located upstream of gene 71 which are active in an L5 lysogen but which do not require any phage-encoded proteins. In early lytic growth, gene 71 is also transcribed from a promoter, Pleft, located at the right end of the genome and which appears to be a target of gp71 regulation. A model is given for the regulation of L5 life cycles.

Comparative in vitro activities of azithromycin, Bay y 3118, levofloxacin, sparfloxacin, and 11 other oral antimicrobial agents against 194 aerobic and anaerobic bite wound isolates.

The activities of sparfloxacin, levofloxacin, Bay y 3118, azithromycin, cefprozil, loracarbef, and nine other oral antimicrobial agents against 194 aerobic and anaerobic clinical bite wound isolates were determined by the agar dilution method. Sparfloxacin, levofloxacin, and Bay y 3118 were active against all aerobic isolates (MICs at which 90% of the isolates are inhibited [MIC90], < or = 1.0 microgram/ml for sparfloxacin and levofloxacin and 0.1 microgram/ml for Bay y 3118) and many anaerobic isolates, with the exception of the fusobacteria. Azithromycin was more active than erythromycin by 1 to 2 dilutions against many aerobes, including Pasteurella multocida and Eikenella corrodens, and by 2 to 4 dilutions against anaerobic isolates. Cefprozil was more active (MIC90, < or = 1 microgram/ml) than loracarbef (MIC90, < or = 4 micrograms/ml) against aerobic gram-positive isolates, but both had poor activity (MIC90, > or = 16 micrograms/ml) against peptostreptococci. Both cefprozil and loracarbef had MIC90s of < or = 0.5 micrograms/ml against P. multocida.

Bacteriophages as tools for vaccine development.

The construction of live recombinant bacterial vaccines requires a reasonably sophisticated genetic system for the introduction, stabilization and expression of foreign antigen genes. Bacteriophages offer a rich collection of tools that can be used for vaccine construction, including site-specific integration-proficient vectors, non-antibiotic selectable markers and signals for efficient transcription and translation of foreign genes. We describe the characterization of a temperate phage of the mycobacteria, mycobacteriophage L5, and application of these phage studies for the construction of recombinant BCG vaccines.

Development and evaluation of a blood-free medium for determining growth curves and optimizing growth of Rochalimaea henselae.

Two strains of Rochalimaea henselae were used to optimize a blood-free growth medium. Seven agar bases, four broths, and combinations of eight supplements were evaluated. Acceptable growth was achieved in media containing Fildes solution and hemin, with the best growth demonstrated in brucella broth or on brucella agar with 6 to 8% Fildes solution and 250 micrograms of hemin per ml. R. henselae utilized hemin in concentrations six times that utilized by Rochalimaea quintana. Erythrocyte membrane was necessary to achieve the full growth-promoting effect of rabbit blood.