Loss of transactivation and transrepression function, and not RPA binding, alters growth suppression by p53.

The tumor suppressor protein p53 activates transcription from promoters with specific p53 binding elements, represses transcription from promoters without such elements and interacts with and inhibits the single-stranded DNA binding activity of the human DNA replication factor RPA. All these activities involve the N terminal 70 amino acids of p53. Dissection of the domains of p53 which bind RPA suggest that multiple sub-domains of the protein synergize to give strong RPA binding. Point-mutations in one of these sub-domains of p53 significantly diminish its ability to interact with RPA. A multimer of a peptide from p53 which includes these residues, or of a peptide from the acidic activation domain of the prototypic trans-activator protein VP16, can itself bind to RPA. Comparison of sequences of these multimeric peptides suggests that aromatic amino acids flanked by negatively charged residues are important for binding RPA. Several alleles of p53 with point mutations in the N terminal region were analysed for their relative abilities to bind RPA, activate or repress transcription, and suppress growth of p53 null SaOs2 and H1299 cells. Both mutants of p53 with decreased RPA binding suppressed cell growth as well as wild-type p53, suggesting that p53 can suppress growth without interacting with RPA. The allele that lost most of the transcription activation function also lost most of its transcription repression activity suggesting that interaction with the same basal transcription factors are involved in both functions. This same allele bound RPA well but was defective in growth suppression. Therefore, transcription activation and/or repression appear to be more important for the growth suppression function of p53 than RPA binding.

Cyclins as markers of tumor proliferation: immunocytochemical studies in breast cancer.

We have developed methods to use anticyclin A, B, and E antibodies as reagents to specifically detect proliferating cells in specific phases of the cell cycle in formalin-fixed, paraffin-embedded sections of tissues and cells. Staining of 48 archival cases of breast cancer showed that these antibodies estimate the tumor proliferation fraction and therefore are potentially useful for the prediction of prognosis. A subset of cancers had a high frequency of tumor cells expressing cyclins A and E, out of proportion to other proliferation markers, suggesting that these tumors may have deregulated cyclin expression. In addition to recognizing authentic cyclin E in the nucleus of proliferating cells, anticyclin E antibody cross-reacted with a cytoplasmic protein in nonproliferating endothelial cells. This cross-reaction allows the simultaneous visualization and quantitation of microvessels in the tumors, measuring a second potential predictor of breast cancer prognosis, tumor angiogenesis.

Iron status alters murine systemic lupus erythematosus.

The effect of Fe status on murine systemic lupus erythematosus was investigated. Weanling female MRL/MPJ-lpr/lpr mice (systemic lupus erythematosus strain) were fed diets with the following levels (mg Fe/kg diet): 3 (severely deficient), 10 (moderately deficient), 35 (control) and 250 (supplemented). A fifth group was pair fed the control diet in the amounts consumed by the severely deficient group. C3H/Hej mice fed the same diets were used as non-lupus controls. Anemia was more severe in severely deficient mice than in all other MRL groups and C3H severely deficient mice. Incidence of skin lesions was highest in MRL severely and moderately deficient mice compared with pair-fed, control and supplemented mice. By 22 wk of age, mortality was higher in supplemented and severely deficient mice than in moderately deficient, pair-fed and control MRL mice. Anti-dsDNA activity in serum was not altered by Fe. In a second experiment, kidney function was examined in mice fed severely deficient, control, supplemented and pair-fed diets. Urine protein concentration was highest in supplemented mice at 14 wk of age. Serum urea nitrogen was significantly higher in MRL severely deficient mice than in pair-fed and control mice at 18 wk of age. Glomerular filtration rate, measured by creatinine clearance, was significantly lower in MRL severely deficient mice than in pair-fed and Fe supplemented mice at 16 wk of age and pair-fed and control mice at 18 wk of age. Renal histopathology was more severe in Fe supplemented mice than in pair-fed and control mice, and more severe in severely deficient and pair-fed mice than in control mice. Fluorescent staining of kidneys with anti-Ig G and anti-C3 fluorescein-conjugated antibodies was most intense in severely deficient mice, and the concentration of circulating immune complexes in serum was significantly higher in severely deficient mice than in all other groups. These data demonstrate that systemic lupus erythematosus in MRL/MPJ-lpr/lpr mice is altered by dietary iron.

Chloramphenicol-induced mitochondrial dysfunction is associated with decreased transferrin receptor expression and ferritin synthesis in K562 cells and is unrelated to IRE-IRP interactions.

Chloramphenicol is an antibiotic that consistently suppresses the bone marrow and induces sideroblastic anemia. It is also a rare cause of aplastic anemia. These toxicities are thought to be related to mitochondrial dysfunction, since chloramphenicol inhibits mitochondrial protein synthesis. We hypothesized that chloramphenicol-induced mitochondrial impairment alters the synthesis of ferritin and the transferrin receptor. After treating K562 erythroleukemia cells with a therapeutic dose of chloramphenicol (10 microg/ml) for 4 days, there was a marked decrease in cell surface transferrin receptor expression and de novo ferritin synthesis associated with significant decreases in cytochrome c oxidase activity, ATP levels, respiratory activity, and cell growth. Decreases in the transferrin receptor and ferritin were associated with reduced and unchanged message levels, respectively. The mechanism by which mitochondrial dysfunction alters these important proteins in iron homeostasis is not clear. A global decrease in synthetic processes seems unlikely, since the expression of the cellular adhesion proteins VLA4 and CD58 was not significantly decreased by chloramphenicol, nor were the message levels of beta-actin or ferritin. The alterations were not accompanied by changes in binding of the iron response protein (IRP) to the iron-responsive element (IRE), although cytosolic aconitase activity was reduced by 27% in chloramphenicol-treated cells. A disturbance in iron homeostasis due to alterations in the transferrin receptor and ferritin may explain the hypochromic-microcytic anemia and the accumulation of nonferritin iron in the mitochondria in some individuals after chloramphenicol therapy. Also, these studies provide evidence of a link between mitochondrial impairment and iron metabolism in K562 cells.

Translation of the alzheimer amyloid precursor protein mRNA is up-regulated by interleukin-1 through 5'-untranslated region sequences.

The amyloid precursor protein (APP) has been associated with Alzheimer's disease (AD) because APP is processed into the beta-peptide that accumulates in amyloid plaques, and APP gene mutations can cause early onset AD. Inflammation is also associated with AD as exemplified by increased expression of interleukin-1 (IL-1) in microglia in affected areas of the AD brain. Here we demonstrate that IL-1alpha and IL-1beta increase APP synthesis by up to 6-fold in primary human astrocytes and by 15-fold in human astrocytoma cells without changing the steady-state levels of APP mRNA. A 90-nucleotide sequence in the APP gene 5'-untranslated region (5'-UTR) conferred translational regulation by IL-1alpha and IL-1beta to a chloramphenicol acetyltransferase (CAT) reporter gene. Steady-state levels of transfected APP(5'-UTR)/CAT mRNAs were unchanged, whereas both base-line and IL-1-dependent CAT protein synthesis were increased. This APP mRNA translational enhancer maps from +55 to +144 nucleotides from the 5'-cap site and is homologous to related translational control elements in the 5'-UTR of the light and and heavy ferritin genes. Enhanced translation of APP mRNA provides a mechanism by which IL-1 influences the pathogenesis of AD.