Upregulation of endogenous p53 and induction of in vivo apoptosis in B-lineage lymphomas of E(mu)-myc transgenic mice by deregulated c-myc transgene.

E(mu)-myc transgenic mice carry a constitutively overexpressed c-myc oncogene and develop B-lineage lymphomas. Previous studies have shown that c-myc overexpression can lead to in vitro apoptosis. Here, we investigated the in vivo effects of altered c-myc expression on cell proliferation versus death in spontaneously arising E(mu)-myc tumors. E(mu)-myc tumors display extensive in vivo apoptosis confined to small clusters of cells with greatly increased expression of both the c-myc transgene and the endogenous p53 gene as compared with that in normal, pretumor, or surrounding tumor tissue. This restricted overexpression of both the c-myc transgene and the endogenous p53 gene in small clusters of apoptotic tumor cells indicates that overexpression of these genes and apoptosis are not obligatory or uniform during tumor development and suggests that further somatic mutations or microenvironmental influences may be responsible for these properties. Nevertheless, the clear ability of tumor cells to undergo apoptosis in vivo may be exploitable for therapeutic purposes.

Relationship between expression of epidermal growth factor and simian virus 40 T antigen in a line of transgenic mice.

The pattern of expression of the simian virus 40 (SV40) T antigen gene and resultant dysplasia were re-examined in a line of transgenic mice in which the T antigen gene was under the control of the SV40 early promoter. We found that T antigen expression in the kidney, and resulting dysplastic lesions, occurred exclusively in the distal convoluted tubules and the ascending limbs of Henle. Epidermal growth factor (EGF) expression in the kidney of normal mice was similarly immunolocalized. The correlation between high EGF immunoreactivity in normal mouse tissues and T antigen expression in the transgenic counterpart was also seen in the choroid plexus epithelium and in the submandibular glands of male mice. T antigen was not found in the submandibular gland of transgenic females. Similarly, EGF was only rarely detected in the normal female submandibular gland. In contrast to the correlation between T antigen expression in the transgenic mice and EGF expression in the corresponding tissues of the normal mice, within the dysplastic lesions of the transgenic mice EGF expression was severely diminished. Adenocarcinomas of the male submandibular gland from another line of transgenic mice that expresses the Int-1 transgene, showed similarly reduced levels of immunostaining for EGF. Thus, reduced expression of EGF might be a general feature of dysplasia and tumorigenesis in those tissues that normally express EGF.

Autoantibodies to c-myc protein: elevated levels in patients with African Burkitt's lymphoma and normal Ghanians.

Sera from U.S. patients with SLE, RA, and various malignancies, clinically normal individuals with sero-activity to HIV, AIDS, and from pregnant women were tested for the presence of anti-c-myc antibodies. In an ELISA using recombinant human c-myc protein as the antigen, no difference in mean antibody titer was generally detected in these sera when compared to normal controls. Only three malignancy sera (two myeloid leukemia and only one lymphoma) and two patients with AIDS-related lymphoma exhibited exceedingly higher levels of anti-c-myc antibody. However, significantly elevated anti-c-myc antibody levels were found among 20 patients with African Burkitt's lymphoma (Ghana) and 20 normal Ghanians, thus apparently reflecting an autoimmune phenomenon prevalent in the endemic region. These findings indicated that elevated levels of anti-c-myc antibodies are not a general characteristic of patients with diseases that have been associated with increased expression of c-myc.

Immunocytochemical localization of glucocorticoid receptors in cells, cytoplasts, and nucleoplasts.

A monoclonal antibody has been used to assess the intracellular localization of the glucocorticoid receptor in rodent L-929 fibroblasts and GH3 pituitary tumor cells. Whole cells from both cell lines showed immunoreactivity in the cytoplasm and nucleus. However, when cytoplasts and nucleoplasts of these cells were examined, only L-cells showed strong antibody binding in both fractions; in contrast, GH3 cells exhibited nuclear staining and slight cytoplasmic staining. These results are discussed in terms of the current findings regarding the intracellular location of steroid hormone receptors.

Reactivation of avian erythrocyte nuclei in mammalian cytoplasts. A dominant role for pre-existing cytoplasmic components.

Antibodies and inhibitors have been used to study the process of nuclear reactivation following the fusion of chick erythrocytes with mouse L cell cytoplasts. Immunofluorescence results showed that a monoclonal antibody against a DNA 'tight-binding' protein from HeLa chromatin as well as an anti-Sm human serum failed to bind to the unreactivated erythrocyte nucleus, but showed strong binding after fusion. The development of antibody-binding sites was affected neither by alpha-amanitin nor by cycloheximide, indicating that some of the processes of reactivation, including specific protein uptake are independent of DNA and RNA synthesis. These results are discussed in terms of the role of the chick nucleus in directing the reactivation process.

Status of the nuclear matrix in mature and embryonic chick erythrocyte nuclei.

The adult chicken erythrocyte nucleus was found to lack an internal nuclear matrix: even milder extraction procedures resulted in the production of empty shells of pore complex-lamina together with loose aggregates of core histone. In contrast, rat liver nuclei showed a typical intranuclear salt-resistant skeleton. These results show that an internal matrix is not an obligatory nuclear component, and is not required for the spatial organization of chromatin. 5-day-old embryonic erythrocytes did, however, contain an interchromatinic nuclear matrix, suggesting a correlation between the presence of matrix structures, and nuclear 'activity'.

Generation of an internal matrix in mature avian erythrocyte nuclei during reactivation in cytoplasts.

When fused with mouse L-cell cytoplasts, chick erythrocyte nuclei enlarge, take up proteins from the host cytoplasm, and recommence RNA synthesis. We found that during this transition the erythrocyte nuclei gain an internal nuclear matrix, thus providing a novel approach to questions concerning the nature of the salt-resistant intranuclear skeleton. A new method for preparation and examination of the nuclear matrix in situ is also described.

Micrococcal nuclease cleavage of chromatin displays nonrandom properties.

A statistical analysis of the products of digestion of chicken erythrocyte chromatin by micrococcal nuclease was used to test for randomness of the cutting process. DNA fragment size classes corresponding to mononucleosome, dinucleosome, trinucleosome, tetranucleosome, and all fragments larger than tetranucleosome were evaluated. In every case, fragments in the mononucleosome and greater-than-tetranucleosome classes were produced in excess of the level expected on the basis of random cleavage while those in the dinucleosome-tetranucleosome classes exhibited a shortage. The pattern of nonrandomness appears to depend on substrate size: the magnitude of deviations from randomness was large when substrates of genomic size are compared with polynucleosomal segments whereas the direction of deviation is identical. Nonrandomness was independent of ionic conditions known to affect the state of chromatin condensation and also appeared to be unaffected by depletion of histones H1 and H5. The possible universality of nonrandom cleavage was suggested when other data from the literature was analyzed. Some possible mechanisms to account for this property are discussed.