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Biomedical subjects

D Mercola

Publications and source records attributed to D Mercola.

At least 55 records · Page 3Linked to original sources
PubMed1995

Antisense approaches to cancer gene therapy.

Recent advances in the use of oligodeoxynucleotide and plasmid-derived RNA as antisense agents of special relevance to cancer gene therapy are summarized with emphasis on agents and systems which have lead to clinical trials and/or regression of established tumors in animal model systems. Transformed cell lines bearing plasmids and viruses designed for the transcription of antisense RNA have the advantage that they can be characterized thoroughly and the effects of antisense RNA on target gene expression and phenotype can be studied easily in vivo. Promising results make the considerable efforts of applying oligodeoxynucleotides in whole animals and in clinical trials more plausible. Conversely, oligodeoxynucleotide experiments which yield promising results in tissue culture can be generalized to the in vivo setting by development of clones of cells bearing plasmid-derived antisense RNA against the same target. Several examples of the concordant results for oligodeoxynucleotide and plasmid-derived antisense RNA against the same target are considered. The importance of examination of antisense effects in syngeneic and immunocompetent hosts is illustrated by studies of insulin-like growth factor and insulin-like growth factor receptor where tumor regression and protection against tumor formation have been observed for particular cell types in defined settings.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Transformation-specific pattern of phosphorylation of c-Jun, Jun-B, Jun-D and Egr-1 in v-sis transformed cells.

The degree of phosphorylation of c-Jun, Jun-B, Jun-D and Egr-1 transcription factors was examined during normal growth and during a prolonged period of defined transformation of NIH-3T3 cells which conditionally express v-sis [Mercola, D. et al. (1992) Oncogene, 7, 1793-1803]. During the asynchronous growth of normal cells phosphorylation of all factors was low and constant at all stages of growth from low density (c. 25 x 10(3) cells/cm2) through log-phase of growth to saturation density (c. 100 x 10(3) cells/cm2). Upon induction of v-sis, a marked and coordinate increase in phosphorylation occurred for c-Jun, Jun-B and Egr-1 to approximately 320%, 230% and 420% respectively above basal levels which was stable for the 2.5 day transformation period. The phosphorylation of Jun-D increased to over 600% and, after about 20 h, steadily declined to near basal levels at 54 h post-induction. Moreover, at any time phosphorylation and v-sis expression were fully reversible upon removal of the inducer. It appears that increased phosphorylation of the Jun family members and Egr-1 is not necessary for normal growth of NIH-3T3 but is dependent upon the expression of v-sis. Thus, normal and transformed cells may be distinguished. For c-Jun, the v-sis enhanced phosphorylation occurs at serines 63 and 73 and is required for transformation by several oncogenes [Smeal, T. et al. (1992) Mol. Cell. Biol., 12, 3507-3513]. The results described here show that the phosphorylation of additional factors is a stable and specific correlate of transformation which have have regulatory significance during transformation.

3T3 Cells↗
PubMed1994

Suppression of v-sis-dependent transformation by the transcription factor, Egr-1.

The transcription factor Egr-1, stimulates the activity of a number of genes and inhibits other genes, by binding to the sequence GCGGGGGCG in 5' enhancer regions. However, the functions of Egr-1 are obscure in spite of its rather ubiquitous expression. Egr-1 may play a role in proliferation in mitogen-stimulated cells but its expression is also correlated with the differentiated state in teratocarcinoma cells. The constitutive expression of Egr-1 appears to have little effect on the growth rate of normal immortalized cell-lines. We show that in NIH3T3 cells that are conditionally transformed by the expression of v-six, the presence of Egr-1 is inhibitory to the production of transformed colonies (foci) and to growth in soft agar. In addition, the first appearance of tumors in nu/nu mice is delayed in tumorigenicity tests with cells that over-express Egr-1 and tumor growth is suppressed compared to control cells. We used a series of fragments of Egr-1 cloned into expression vectors to show that not only full length, but also truncated Egr-1 fragments inhibit colony formation. Using deletion mutants, we observed that this inhibitory activity is dependent on the presence of the DNA-binding 'zinc-finger' region. Wilm's tumor protein, WT1, (known to be a tumor suppressor gene) that exhibits the same DNA binding activity is also inhibitory. In contrast, colony formation is stimulated by an Egr-1 antisense RNA-expressing plasmid, since colonies grow rapidly and the colony-forming frequency is higher than in the presence of v-sis alone. We conclude that proteins containing the Egr-1 'zinc-finger' domain can bind to the regulatory regions of one or more genes that are required for the transformation of fibroblasts by v-sis thus inhibiting transformation. One function for Egr-1 implied by these results is the restraint of transformed growth in mitogen-stimulated cells.

3T3 Cells↗
PubMed1992

Oncoprotein-mediated signalling cascade stimulates c-Jun activity by phosphorylation of serines 63 and 73.

In resting cells, c-Jun is phosphorylated on five sites. Three of these sites reside next to its DNA binding domain and negatively regulate DNA binding. In response to expression of oncogenic Ha-Ras, phosphorylation of these sites decreases, while phosphorylation of two other sites within c-Jun's activation domain is greatly enhanced. Phosphorylation of these residues, serines 63 and 73, stimulates the transactivation function of c-Jun and is required for oncogenic cooperation with Ha-Ras. We now show that the same changes in c-Jun phosphorylation are elicited by a variety of transforming oncoproteins with distinct biochemical activities. These oncoproteins, v-Sis, v-Src, Ha-Ras, and Raf-1, participate in a signal transduction pathway that leads to increased phosphorylation of serines 63 and 73 on c-Jun. While oncogenic Ha-Ras is a constitutive stimulator of c-Jun activity and phosphorylation, the normal c-Ha-Ras protein is a serum-dependent modulator of c-Jun's activity. c-Jun is therefore a downstream target for a phosphorylation cascade involved in cell proliferation and transformation.

3T3 Cells↗
PubMed1992

Rapid, complete and reversible transformation by v-sis precedes irreversible transformation.

v-sis is the oncogene of simian sarcoma virus, but whether tumor growth is maintained by v-sis expression alone or requires additional changes is unknown. To distinguish these possibilities we studied a model of reversible transformation including tumorigenicity using NIH3T3 cells bearing a metallothionein promoter-v-sis construction. Cells subcultured from 10 out of 18 tumors from athymic mice, all less than 0.1 g and less than or equal to 21 days in age, reverted to a normal phenotype but exhibited transformation upon addition of zinc as judged by morphology, growth rate, saturation density and anchorage independence of growth. Thus, activation of v-sis alone is sufficient for initiation and early autocrine-based growth of tumors. However, the cells from the remaining and predominantly larger, 0.5 +/- 0.7 g, tumors did not revert and exhibited zinc-independent transformation as judged by the same criteria. Southern analysis and examination of the regulation of v-sis product expression in cells derived from these tumors showed no change in zinc-dependent and reversible regulation of v-sis sequences. These results suggest that subsequent tumor growth strongly favors acquisition of additional irreversible change(s) in the tumor cell genome at high frequency (44%). Thus an early event of a multistep process stimulated by v-sis-dependent transformation best accounts for the sum of results.

3T3 Cells↗
PubMed1991

Reversible transformation by v-sis: a model cell line for analysis of transformation by antisense methods.

Cellular homologs of v-sis are implicated in numerous human tumor types (Eva et al., 1982; Betsholtz et al., 1984; Johnson et al., 1985; Bronzert et al., 1987; Igarashi et al., 1987; Nister et al., 1988a,b; Versnel et al., 1988; Matsui et al., 1989), but whether tumor growth is maintained by sis expression alone or requires additional changes is unknown. To distinguish these possibilities, we studied reversible transformation of NIH-3T3 cells bearing an inducible v-sis construction. Cells subcultured from 10 of 18 tumors, all less than 0.1 gram and less than or equal to 21 days in age, reverted to a normal phenotype by four criteria, but again exhibited transformation upon induction. Thus, activation of the v-sis autocrine loop alone is sufficient for initiation of tumors. Cells from the remaining and larger, 0.5 +/- 0.7 gram, tumors did not revert by any criteria. This suggests that subsequent tumor growth is maintained by acquisition of irreversible change(s) that occurs at high frequency in vivo.

3T3 Cells↗
PubMed1990

The proto-oncogene c-fos is over-expressed in the majority of human osteosarcomas.

The expression of fos protein was examined in 30 cases of human osteosarcoma as formalin-fixed and paraffin-embedded tissue sections using two monoclonal antibodies and, for five cases, as frozen sections using 3 polyclonal antibodies. Nuclear antibody labeling intensities were determined either by visual scoring (3 pathologists) or by microdensitometry and included over 700 tumor, 350 normal and 150 benign tissue observations. Visual scores were shown to be linear with optical density with a correlation coefficient of 0.97. Analysis of the osteosarcoma cases at one antibody concentration revealed two groups: a minority (39%) with a low average visual score of 2 +/- 0.2 which was very similar to benign and normal tissues, and a majority (61%) with an average score of 3.1 +/- 0.3. The difference is highly significant (t-test), P less than or equal to 0.01. The results were supported by an analysis of partial immunotitration curves using a curve-fitting procedure which yielded estimates of FOSo (maximum relative fos protein concentration) which were ca. 150% increased for the majority tumor group compared to the minority group or benign or normal tissues. In previous studies of v-sis transformed cells, which exhibit ca. 300% over-expression of c-fos protein as observed here, antisense techniques were used to show that over-expression leads to increased growth and loss of contact inhibition. Thus the combined results suggest that steady state c-fos protein is significantly elevated in, and may contribute to, the aggressive growth properties of a majority of human osteosarcomas.

Antibodies, Monoclonal↗
PubMed1988

Analysis of a transformed cell line using antisense c-fos RNA.

Simian sarcoma virus (SSV)-infected NIH-3T3 cells (SSV-NIH-3T3), express a homologue of platelet-derived growth factor, (PDGF) a powerful inducer of the c-fos gene. We have used these cells to test the hypothesis that autocrine stimulation by PDGF-like molecules leads to c-fos expression which is functional in the transformed phenotype. We have transfected SSV-NIH-3T3 cells with a c-fos antisense-RNA expression vector, pSVsof, or control plasmids. pSVsof-transfected cells exhibit markedly decreased c-fos mRNA and protein levels, restored density-dependent growth arrest and reduced (three of five clones) tumorigenicity compared to control lines. The results confirm that c-fos cooperates in the transformed phenotype of SSV-NIH-3T3 cells.

Animals↗
PubMed1979

Interaction of troponin subunits. The interaction between the inhibitory and tropomyosin-binding subunits.

The interaction of troponin-I and troponin-T was demonstrated by circular dichroism and gel filtration. Troponin-I gives a negative circular dichroism band between 300 and 260 nm while troponin-T gives two weak positive bands, one at 290 nm and the other at 263 nm. When troponin-I and troponin-T were mixed, the complex produced a strong negative circular dichroism band with a maximum around 280 nm. This band was most intense with a molar ratio of troponin-T to troponin-I of 1:1. The intensity of the band was 2.4 times that expected from the separate components. The interaction was independent of salt concentration from 0.15 to 0.5 M KCl. Gel filtration on Sephacryl S-200 showed that a stable 1:1 complex was formed between troponin-T and troponin-I. When troponin-C was added to the complex of troponin-T.troponin-I; the reconstituted troponin had a circular dichroism spectrum identical to that of native troponin. The oxidation state of troponin-I was important in reconstituting troponin. Oxidized troponin-I produced less change in the near ultraviolet circular dichroism when added to troponin-T and the troponin-C than did reduced troponin-I. This showed the subunits were not assembled correctly with oxidized troponin-I. When the reconstituted complex was reduced, the circular dichroism was restored to that of native troponin. Troponin reconstituted with oxidized troponin-I did not confer calcium sensitivity on actomyosin ATPase; activity was restored by reducing the complex.

Circular Dichroism↗
PubMed1978

Comparison of the calcium- and magnesium-induced structural changes of troponin--C. A proton magnetic resonance study.

Previous proton magnetic resonance studies of the effects of Ca(II) on the structure of rabbit skeletal muscle troponin--C have shown that Ca(II) binding to the two high affinity sites of troponin--C both directs and stabilizes the folding of much of the structure. Ca(II) binding by the two low affinity sites of troponin--C causes changes in the environment of largely hydrophobic residues. We have now examined the structural changes caused by Mg(II) and by Ca(II) in the presence of excess Mg(II). Successive addition of Mg(II) to metal-free troponin--C leads to broadly similar, but not identical, structural changes to those previously assigned to Ca(II) binding at the high affinity sites. None of the changes previously assigned to Ca(II) binding to the low affinity sites was observed. Since Mg(II) does not bind to the low affinity sites, these results confirm the previous assignments. The spectral differences between Mg(II) and Ca(II) show that the degree of backbone folding and interactions between a group of hydrophobic residues (one or more Val, Leu, Ile; two or more Phe) are different for the two cations. In the presence of excess Mg(II), at a molar ratio that may exist in vivo (approx. 40 : 1 mol ratio Mg(II): Ca(II), titration with Ca(II) leads to a displacement of Mg(II) and to all the structural changes previously observed for Ca(II) alone. However, in the presence of Mg(II) the distinction between high and low affinity sites is blurred as judged by the overlap of the spectral changes associated with each of the binding sites. This result, together with the observation that Mg(II) promotes structural changes different from Ca(II), suggests a structural basis for the observation that the Ca(II) threshold for the activation of tension in some myofibrils is increased in the presence of high Mg(II) concentrations.

Animals↗
PubMed1976

Near-ultraviolet tyrosyl circular dichroism of pig insulin monomers, dimers, and hexamers. Dipole-dipole coupling calculations in the monopole approximation.

The tyrosyl circular dichroism (CD) has been calculated using the conformation of pig insulin observed in rhombohedral crystals containing 2 zinc atoms per hexamer. These calculations predict that the tyrosyl CD at 275 nm will be enhanced disproportionally as monomers interact to form dimers and as dimers interact to form hexamers. This enhanced tyrosyl CD (delta epsilon per 5800 molecular weight) results from new coupling interactions generated in the regions of contact between monomers and between dimers. These calculations illustrate that a large CD enhancement may accompany aggregation even in the absence of a conformation change in eith monomer. The tyrosyl CD intensities calculated for monomers, dimers, and hexamers of 2-zinc pig insulin are compatible with the experimentally observed CD spectra which are enhanced about fourfold in the hexamer compared with the monomer. Zinc ions and other metals do not contribute directly to the tyrosyl CD but only influence the optical properties by promoting the hexameric state. The relation of the integrity of the molecule to dimer formation and the biological activity of the molecules are discussed. The largest calculated contributions to tryosyl CD arise from interactions with far-ultraviolet transitions of neighboring aromatic groups. In the hexamer, about half of the tyrosyl CD intensity is calculated to arise from Tyr-A14.

Animals↗