Induced Differentiation of Epithelioid Carcinoma Cell Lines: Evidence for Tumor Cell Quantal Mitosis

The effects of growth factors and calcium concentrations present in different culture media on induction of terminal differentiation were investigated for four different epidermoid carcinoma cell lines, Hela, KB, A431, and SCC-25, and their responses determined relative to those elicited by normal human keratinocytes subjected to these culture conditions. Differentiation status was determined cyto-chemically by a validated keratin protein staining method, and by autoradiographic analyses. Growth and differentiation promoting factors that influenced the direction of integrated control of growth and differentiation in normal human keratinocytes were found to be effective for some cell lines but not others. The factors examined were 1) high density arrest in serum-free and serum-containing media, 2) media shifts from high density culture in serum-containing media to low density growth factor-depleted or supplemented serum-free medium, and 3) the concentration of calcium in the media. The extent and degree of differentiation achieved varied among different cell lines depend on the presence or absence of serum, EGF and insulin protein growth factors. Certain growth media appear to sponsor keratin protein, cyto-chemically-detected differentiation, and evidence of quantal mitotic division in low density HeLa cell and SCC25 cell cultures. Epidermoid carcinoma cell lines retain limited capacity to commit to early stages of cell differentiation.

Cancer Chemopreventive Retinoids: Validation and Analysis of in Vivo and in Vitro Bioassay Results

Several natural and synthetic retinoids (vitamin-A derived analogies) were examined for their potential anti-cancer activity in both in vivo animal models and a novel in vitro human keratinocyte clonal growth bioassay system. The natural retinoids included all-trans-retinoic (RA), 13-cis-retinoic acid, 4-oxoretinoic acid, and retinol. Among the synthetic retinoids tested were all trans N-(4-hydroxy(phenyl)retinamide, 3-substituted oxoretinoic acids, and 13 cis-N-ethylretinamide. The animal models employed were: 1) vitamin A-deficient hamster tracheal organ assay (HTOC);2) the benzo(α)pyrene-induced squamous metaplasia in a hamster tracheal organ system (BP-HTOC);3) the mouse skin tumor promoter (TPA)-induced ornithine decarboxylase enzyme assay(ODC);4) the mouse skin papilloma (MPA) assay;and 5) a novel retinoid bioassay in which retinoids display IC50 values to inhibit clonal growth of NHK. All-trans-RA, 4-oxoretinoic acid and retinol were consistently more active than any of the synthetic derivatives in all bioassays tested. A statistical model was developed and significant positive correlations were found between: 1) ED50 values in the HTOC system and reduction in TPA-induced ODC enzyme activity;2) tumors per animal in the MPA bioassay and suppression of TPA-induced ODC activity;and 3) a positive correlation between suppression of tumors per animal in the MPA assay, and retinoid inhibition of keratinocyte clonal growth. Test retinoids, were tested for their capacity to inhibit the clonal growth of a squamous carcinoma cell line (SCC-25), which were found to be 2 - 3 logs less sensitive for each tested retinoid than the corresponding activity against NHK cells. Antineoplastic retinoid drugs were reviewed.

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.

Differential Anticancer Effect of an Apple Extract (Applephenon^&reg), Polyphenols and Isoflavones on Normal Human Keratinocytes and Epidermoid Cancer Cells

Applephenon&reg, a purified extract prepared from green apples, was examined for its cytotoxicity and inhibitory effects on the proliferation of cultures of normal human keratinocytes and several epidermoid cancer cell lines. Our HPLC studies demonstrated a high content of phenolic compounds (>65%), including catechin, epicatechin, caffeic acid and phloretin as well as polyphenols such as proanthocyanidins. Applephenon&reg demonstrated a greater cytotoxic effect against HeLa, A431 cancer cell lines and HaCaT, an immortalized keratinocyte cell line than serum-free cultures of proliferating normal human keratinocytes (NHK). Proliferation of NHK was inhibited at concentrations above 0.0013% while concentrations above 0.005% were cytotoxic. By contrast, Applephenon&reg solutions above 0.00025% killed each of the cancer cell lines. Treated cells displayed increased intercellular separation and evidence of keratinizing stratification. We also tested the effect of epicatechin, and two isoflavonoids, genistein and daidzein, on cancer cell lines. Hela cells were more sensitive to epicatechin and genistein inhibition of cell growth and cytotoxicity than were NHK. Daidzein at these concentrations had little effect on cancer cells. These results indicate that Applephenon&reg and some of its phenolic components have selective anticancer activity.

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.

Evidence for pentagonal symmetry in living and model cellular systems

Microscope observations of normal human ke- ratinocytes (NHK) propagated in a serum-free medium reveal a high frequency (>70%) of pentagonally-shaped colonies over a wide range of colony sizes that persist over many sequential cell generations. NHK colonies derived from sin- gle cell isolates also display pentagonal symme- try as confirmed by a photographic technique known as “Markham Rotation”. The generality of pentagonal cellular morphology was extended to observations in situ of pentagonally-shaped basal layer epidermal cells of normal human epidermis, monolayer cultures of normal and immortalized keratinocytes, several different ch- ick embryo cells, and in previously published photographs. Statistical methods were applied that differentiate planar close-packing of polygonal configurations observed in living cellular system from several examples of non-living cellular aggregates that were produced spontaneously in nature or in the laboratory under defined physico-chemical conditions.

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.