Effects of Okadaic Acid, Retinoic Acid, and Phorbol Myristate Acetate Tumor Promoter on Oncogene Expression

The effect of okadaic acid (OA) on proto-oncogene protein expression of c-neu, c-myc, v-rasH, EGFR, and phosphotyrosine-containing phosphoproteins (P-Tyr) was investigated in rapidly growing (RG) normal human keratinocytes (NHK) and in SV-40 virally-transformed keratinocytes (SVK) cultured in a growth factor supplemented serum-free medium as assessed by indirect immunofluorescence microscopy. P-Tyr positively stains cell surface antigens (cytoplasm) diffusely at monopolar sites in RG NHK cultures. OA-treatment intensifies cytoplasmic P-Tyr staining at localized monopolar intercellular focal adhesion (IFA) sites with reduced cytoplasmic staining. P-Tyr expression was predominate at IFA sites with little cytoplasmic staining in RG SVK cultures. OA-treatment increased monopolar P-Tyr staining and cytoplasmic staining. OA-treatment in RG NHK cultures intensified cytoplasmic staining of c-myc and EGFR (epidermal growth factor receptor) expression. OA-treatment in RG NHK and SVK cultures intensified c-neu staining at monopolar IFA sites and intensified c-neu staining at both cytoplasmic and bipolar IFA sites in RG SVK cells. OA was especially cytotoxic for SVK cells. RA treatment decreased c-neu expression in RG NHK cultures while TPA treatment has a lesser effect on both cytoplasmic and IFA sites. RA treatment also decreased P-Tyr staining in both NHK and SVK cells. Again, TPA had a lesser inhibitory effect on P-Tyr staining pattern. RA-treatment had a similar effect on P-Tyr staining of RG cultures of a mouse fibroblast cell line. These results confirm the generality of OA, RA and TPA on the regulation of oncogene expression in both normal and malignantly transformed keratinocytes.

Occurrence of Fibonacci numbers in development and structure of animal forms: Phylogenetic observations and epigenetic significance

A survey of zoological literature affirmed the wide occurrence of Fibonacci numbers in the organization of acellular and prokaryotic life forms as well as in some eukaryotic protistans and in the embryonic development and adult forms of many living and fossil remains of metazoan animals. A detailed comparative analysis of the axial skeleton of a fossil fish and humans revealed a new rule of the “nested triad” of bones organized along the proximal to distal axis of limb appendages. This growth pattern and its ubiquity among living vertebrates appear to underlie a profound rule of pattern formation that is dictated in part by the genetics and epigenetic mechanisms of stem cell clonal development.

Cell Cycle Arrest Mediates Global DNA Methylation Patterns in Normal Human Keratinocytes, Epidermoid Carcinoma Cells and Murine Embryonic Fibroblasts

The 5-methylationcytosine (5-MC) DNA content of murine embryonic fibroblasts arrested in G1 by four growth conditions (Gc, Gn, Gd, and Gs) were hypermethylated relative to rapidly growing (RG) fibroblasts. Normal human keratinocytes (NHK) arrested in G1 by suspension were hypermethylated relative to RG cultures. Four RG cultures of epidermoid carcinoma cells (ECC) were hypomethylated relative to RG NHK cultures, and two cultures (SCC25 and A431) were further hypomethylated by SUS-induced arrest. Linear regression analyses established a positive linear correlation between growth rate and 5-MC content for three murine fibroblasts lines, and a negative correlation for both NHK and ECC lines.

Retinoid and Ethanol-Sensitive Benzo(<i>α</i>)Pyrene Induction of Cytochrome P450 in Human Keratinocytes

Polycyclic aromatic hydrocarbons (PAHs) induce cytochrome P-450 monoxygenase enzymes that catalyze the formation of DNA adducts. We investigated the effects benzo(α)pyrene (B[α]P) alone or in combination with ethanol on normal human keratinocyte (NHK) growth, induction of cytochrome P-4501A1 (CYP1A1), and modulation of these treatments by retinoic acid (RA) in a serum-free culture medium. Growth-arrested confluent NHK serum-free cultures were treated with B[α]P alone or in combination with ethanol and RA. The effects on CYP1A1 enzyme activity were investigated. B[α]P treatment alone was not toxic to post-confluent cells;sub-toxic ethanol stimulated cell growth regardless B[α]P treatment. No CYP1A1 activity was detected in control or ethanol-treated NHK cell cultures. B[α]P alone induced CYP1A1 activity, and B[α]P plus ethanol treatment further enhanced B[α]P-induced CYP1A1 activity. Pretreatment with all-trans-RA (t-RA) abolished ethanol enhancement of CYP1A1 activity. There is a synergistic action of ethanol in combination with PAH on induction of P-450 cytochrome enzymes. By contrast, RA reverses ethanol enhancement implying a role for retinoid therapy in counteracting the risk posed by combined alcohol and PAH exposure on epidermal cell carcinogenesis.