Cell proliferation in human cutaneous squamous cell carcinoma.

Squamous cell carcinoma (SCC) of the skin, a fairly slow-growing locally invasive tumor that occasionally matastasizes, was studied in 12 patients in vivo with the use of intralesional tritiated thymidine as a marker for cell proliferation kinetics. Analysis of the cell cycle of the germinative population revealed that the duration of S-phase was 12.5 hours; G1-phase, 28.8 hours; mitotic period (M-phase), 2 hours; G-pahse, 6.9 hours; and the total germinative cell cycle, 50.2 hours. The application of cell kinetics to programming chemotherapy for skin cancer with phase-specific cytotoxins was discussed.

Cell proliferation kinetics in the human hair root.

The kinetics of cell proliferation in the matrix cells of human scalp hair were determined in vivo. Autoradiographic analysis was employed on biopsy specimens taken at specific time intervals after intradermal injection of tritiated thymidine (3H-TdR) into the scalps of volunteer subjects. The durations of the S and G2 phases (Ts and Tg2) were 11.1 and 3.6 hr respectively, obtained from the composite percent labeled mitosis curve. The labeling index of the hari matrix cells was 28.6% +/- 2.2%. The durations of mitosis and of the total cell cycle (Tm and Tgc) were calculated to be 0.8 hr and 38.8 hr respectively. The growth fraction of hair root germinative cells was estimated as 75-100% on the basis of "continuous" 3H-TdR exposure over the duration of the cell cycle. It is noted that the kinetics of human hair root cells are quite similar to the kinetics of psoriatic epidermal cells, suggesting that both tissues are approaching the maximum proliferative rate of keratinogenic epithelial cells.

Decreased thymidine incorporation in circulating leukocytes after treatment of psoriasis with psoralen and long-wave ultraviolet light.

Tritiated thymidine incorporation, a measure of DNA synthesis, was studied in circulating leukocytes from patients with widespread psoriasis who were being treated with photochemotherapy using oral 8-methoxypsoralen (8-MOP) and high-intensity, long-wave ultraviolet light (UVA). Seven of 13 psoriasis patients treated with photochemotherapy demonstrated a significant (p less than 0.05) reduction in leukocyte incorporation of tritiated thymidine immediately after UVA in comparison to incorporation before UVA. None of 10 control subjects treated with UVA alone demonstrated such reduction in leukocyte tritiated thymidine incorporation. Photochemotherapy thus affects circulating blood cells in some patients with psoriasis in addition to its therapeutic effect on epidermal cells. Further investigations are needed to determine the reasons for the differences in susceptibility to inhibition of leukocyte DNA synthesis among patients and the possible long-term consequences of such inhibition.

Abnormal proliferation of uninvolved psoriatic epidermis: differential induction by saline, propranolol, and tape stripping in vivo.

Cell proliferation of the uninvolved psoriatic skin was compared with normal skin of volunteers following stimulation by the intradermal injections of saline or propranolol, or stripping of the stratum corneum with pressure-sensitive tape. Initially, no significant in vivo difference in tritiated thymidine labeling indices were observed between normal and uninvolved epidermal cells. However, 48 hr after each stimulus more DNA synthesizing cells were found in the uninvolved psoriatic than in normal epidermis. Of these 3 stimuli, propranolol was the most effective in distinguishing between uninvolved psoriatic skin and skin from normal control subjects. Thus, uninvolved psoriatic skin appears to possess an abnormal regulation of epidermopoiesis that may be amplified by propranolol injection.

Prostaglandin and DNA synthesis in human skin: possible relationship to ultraviolet light effects.

The effect of prostaglandin E2 (PGE2) on DNA synthesis in human skin was evaluated. PGE2 (1 mug) was infected intradermally into normal buttock skin of 15 volunteers followed by tritiated thymidine for autoradiographic quantitation of DNA synthesizing cells. Controls of normal saline, histamine (50 mug), and lower doses of PGE2 were also injected into 8 of the volunteers. Forty-eight hours after injection of 1 mug and 0.1 mug PGE2 there was a 264% and 62% increase, respectively, in the number of DNA synthesizing epidermal cells/high-power field as compared to saline controls. These differences were statistically significant (p smaller than 0.01). Histamine (50 mug) produced a statistically significant 36% higher labeling index compared to its saline controls (p smaller than 0.05). Many types of skin injury, including ultraviolet light (UVL) irradiation, produce an increase in the number of DNA synthesizing cells about 48 hr after the stimulus. Our findings suggest that PGE, a putative mediator of UVL-induced inflammation, may be one of the chemical mediators for the UVL-induced increase in DNA synthesizing cells. Histamine may also contribute to the increase in DNA synthesizing cells following UVL-induced inflammation.

Cytokinetics and chemotherapy of psoriasis.

The successful treatment of psoriasis with folic acid antagonists during the past 25 years has led to extensive research in the areas of cytokinetics and chemotherapy. In this paper we shall review selected aspects of these topics relevant to the treatment of psoriasis. The effectiveness of methotrexate treatment of psoriasis can be related to both the hyperproliferative cytokinetics of psoriasis and an increased biochemical sensitivity of psoriatic epidermal cells to this drug. Future research goals in chemotherapy of psoriasis include (a) optimizing drug schedules for available drugs; (b) identifying other susceptible biochemical points of selective drug attack; (c) identifying secondary advantages in order to facilitate selective drug action in psoriasis, such as ultraviolet light therapy in combination with a systemic drug; and (d) developing topically effective chemotherapeutic agents. Approaches to research on topical therapy are reviewed with specific reference to animal testing models for psoriasis and percutaneous penetration of topically applied agents.

Mouse vaginal assay for topically effective chemotherapeutic agents.

A number of chemotherapeutic drugs have been subjected to intravaginal testing in mice to measure their local inhibitory activity on DNA, RNA, and protein synthesis in vaginal epithelium. The drugs have been tested at various concentrations and in different vehicles and evaluated by autoradiographic techniques. Systemic absorption of the drugs was monitored by simultaneous study of the gastrointestinal mucosa of the rectum. Methotrexate inhibited deoxyuridine incorporation into DNA in both vaginal and rectal epithelium. Several lipid-soluble analogues of methotrexate were found to have no effect on deoxyuridine incorporation. Nitrogen mustard and emetine have been shown to selectively inhibit DNA and protein synthesis, respectively, without systemic effects. This animal assay system may be useful for predicting the effectiveness of potential drugs for the topical treatment of psoriasis.

Metabolism of estradiol-17ß and estrone in human skin.

The in vitro interconversion of estradiol and estrone was demonstrated in foreskin of the newborn. Conversion of estradiol to estrone was demonstrated in skin of the abdominal wall and in vaginal mucosa.

Cell proliferation in normal epidermis.

A detailed examination of cell proliferation kinetics in normal human epidermis is presented. Using tritiated thymidine with autoradiographic techniques, proliferative and differentiated cell kinetics are defined and interrelated. The proliferative compartment of normal epidermis has a cell cycle duration (Tc) of 311 h derived from 3 components: the germinative labeling index (LI), the duration of DNA synthesis (ts), and the growth fraction (GF). The germinative LI is 2.7% +/- 1.2 and ts is 14 h, the latter obtained from a composite fraction of labeled mitoses curve obtained from 11 normal subjects. The GF obtained from the literature and from human skin xenografts to nude mice is estimated to be 60%. Normal-appearing epidermis from patients with psoriasis appears to have a higher proliferation rate. The mean LI is 4.2% +/- 0.9, approximately 50% greater than in normal epidermis. Absolute cell kinetic values for this tissue, however, cannot yet be calculated for lack of other information on ts and GF. A kinetic model for epidermal cell renewal in normal epidermis is described that interrelates the rate of birth/entry, transit, and/or loss of keratinocytes in the 3 epidermal compartments: proliferative, viable differentiated (stratum malpighii ), and stratum corneum. Expected kinetic homeostasis in the epidermis is confirmed by the very similar "turnover" rates in each of the compartments that are, respectively, 1246, 1417, and 1490 cells/day/mm2 surface area. The mean epidermal turnover time of the entire tissue is 39 days. The Tc of 311 h in normal cells in 8-fold longer than the psoriatic Tc of 36 h and is necessary for understanding the hyperproliferative pathophysiologic process in psoriasis.

Percutaneous absorption of methotrexate: effect on epidermal DNA synthesis in hairless mice.

One of the presumed reasons for the lack of clinical activity of topical methotrexate in psoriasis is insufficient percutaneous penetration necessary to inhibit epidermal DNA synthesis. The present study was undertaken to select a vehicle to optimize penetration of methotrexate in vitro and to determine the effects of this topical formulation on epidermal DNA synthesis in vivo in hairless mouse skin. Increased penetration of methotrexate was obtained in human skin in vitro with Vehicle N compared to water and n-decylmethylsulfoxide vehicles. Repeated topical application of this methotrexate/Vehicle N preparation produced marked epidermal atrophy in treated sites in both normal and hyperproliferative essential fatty acid deficient hairless mouse skin without similar effects at a distant skin site. Local inhibition of epidermal DNA synthesis was also obtained without systemic effects at a distant site. These studies demonstrate that methotrexate in Vehicle N may produce a direct effect on epidermis which may be useful for the topical therapy of psoriasis.

Cell kinetic basis for pathophysiology of psoriasis.

Studies on the cell proliferation kinetics of psoriatic epidermal cells are presented and the results compared to similar studies for normal epidermis. The short 36-h duration of the psoriatic cell cycle (Tc) is confirmed with the first double-peaked fraction of labeled mitoses (FLM) curve in human subjects. The growth fraction of psoriasis using two experimental techniques approximates 100% within 36 h, confirming the rapid Tc found by the FLM method. The cell kinetic basis for the pathophysiology of psoriasis consists of at least 3 proliferative abnormalities in comparison to normal epidermis. By far the largest alteration is the shortening of the Tc from 311 to 36 h. There is also a doubling of the proliferative cell population in psoriasis from 27,000 to 52,000 cells/mm and an increase in the growth fraction from 60% to 100%. As a consequence of these abnormalities the psoriatic epidermis produces 35,000 cells/day from a proliferative compartment of 52,000 cells/mm2 surface area. This is a 28-fold greater production of cells than the 1,246 cells/day produced in normal epidermis. The biochemical or control factors leading to these kinetic differences continue to remain elusive.

Topical tritiated thymidine for epidermal growth fraction determination.

Direct autoradiographic identification of the epidermal growth fraction (GF) requires the delivery of tritiated thymidine ([3H]dThd) to the skin during the time interval of an entire cell cycle. The GF in normal human epidermis has not been directly measured using this technique because the systemic infusion of radioactive [3H]dThd in benign skin conditions is precluded by ethical considerations. Studies were undertaken to assess the feasibility of measuring the epidermal GF in vivo by the topical delivery of [3H]dThd. The percutaneous penetration of [3H]dThd in various vehicles was evaluated to select an effective topical delivery system. A vehicle consisting of Azone, isopropanol, and water (2:49:49) was the best of 4 different vehicles tested. The optimal penetration of [3H]dThd, with respect to the concentration of Azone over a range of 0-4%, was achieved at 2%. During the initial 24 h following a single topical application of [3H]dThd to hairless mice the labeling increased linearly with time. In vivo studies in hairless mice produced a GF of 95% by both continuous systemic [3H]dThd infusion, and by twice daily topical [3H]dThd. Azone vehicles induced epidermal hyperplasia which was minimized by lowering the Azone concentration and by decreasing the frequency of applications from 24 to 48 h. These studies establish the rationale for using topical delivery of [3H]dThd for the in vivo measurement of epidermal GF.

In vitro screening of biochemical activity of folic acid antagonists in skin.

Dihydrofolate reductase (DHFR) inhibitors, which differ from the classical folate antagonists in physicochemical and pharmacologic parameters such as lipid solubility and mechanisms of cellular transport, were screened for DHFR inhibitory activity and biologic activity in newborn rat skin. The most effective drugs from this screen were tested for their effects on de novo DNA synthesis in psoriatic epidermis in vitro. Of the 24 compounds studied, methotrexate (MTX) was the most potent inhibitor of rat skin DHFR (I50=8.6 X 10(-9) M). Methotrexate-dimethylester, methasquin-diethylester, DDEP (2,4-diamino-5-(3',4'-dichlorophenyl)-6-ethylprimidine), and Baker's triazine antifolate (NSC 139105), while less effective than MTX as DHFR inhibitors, were more effective than MTX as inhibitors of de novo DNA synthesis in rat skin in vitro. Baker's antifolate was the only compound tested which was considerably more effective than MTX as an inhibitor of de novo DNA synthesis in psoriatic epidermis in vitro.

Development of a topical hematoporphyrin derivative formulation: characterization of photosensitizing effects in vivo.

Photochemotherapy offers a unique approach for the selective therapy of skin diseases. Hematoporphyrin derivative (HPD) in combination with visible light exhibits cytocidal activity in vitro and systemically has demonstrated applicability to the treatment of experimental and human tumors. This study was undertaken to investigate the phototoxic effects in guinea pig skin of systemic HPD in comparison with locally (intradermal) and topically administered HPD. Maximum erythema was obtained by irradiation with red light or UVA 6 h postsystemic HPD (10 mg/kg). Erythema response was dependent upon the dose of irradiation. Systemic HPD produced complete inhibition of epidermal DNA, RNA, and protein synthesis 6-12 h postirradiation with red light, with a lesser degree of inhibition in the deeper hair roots. Local (intradermal) HPD (5-500 micrograms) in combination with red light or UVA produced a dose-dependent erythema and inhibition of epidermal DNA synthesis. Effective in vitro percutaneous penetration of HPD was demonstrated in vehicles containing Azone and N-methylpyrrolidone. Topical application of these HPD formulations in vivo in combination with red light or UVA produced significant erythema and inhibition of epidermal DNA synthesis. These results suggest that HPD can cause photosensitization of the skin. It may therefore be reasonable to explore topical applications as an alternative approach for the photochemotherapy of psoriasis and other cutaneous diseases.

Regulation of epidermal proliferation in mouse epidermis by combination of difluoromethyl ornithine (DFMO) and methylglyoxal bis(guanylhydrazone) (MGBG).

Topical methylglyoxal bis(guanylhydrazone) (MGBG) and alpha-difluoromethylornithine (DFMO) individually have been shown to produce partial clinical improvement in psoriasis. In an effort to further enhance therapeutic activity, studies were designed to optimize percutaneous penetraton of DFMO in vitro and to determine the effects of the combination of DFMO and MGBG on DNA synthesis and polyamine levels in hairless mouse skin. MGBG was shown to be more effective than DFMO in inhibiting DNA synthesis in vitro and in vivo. Maximum in vitro percutaneous penetration of DFMO (5%) was obtained in Vehicle N containing 10% Azone (297 micrograms/h/cm2). Topical administration of the combination of 5% DFMO and 0.1% MGBG in this vehicle produced a greater inhibition of DNA synthesis and depletion of polyamine levels than either drug individually. The simultaneous topical administration of DFMO and MGBG, allowing the use of lower concentrations of the more toxic MGBG, may be useful for therapy of psoriasis and other cutaneous disorders associated with abnormalities in polyamine metabolism.

Proliferating cells in psoriatic dermis are comprised primarily of T cells, endothelial cells, and factor XIIIa+ perivascular dendritic cells.

Determination of the cell types proliferating in the dermis of patients with psoriasis should identify those cells experiencing activation or responding to growth factors in the psoriatic dermal milieu. Toward that end, sections of formalin-fixed biopsies obtained from 3H-deoxyuridine (3H-dU)-injected skin of eight psoriatic patients were immunostained, followed by autoradiography. Proliferating dermal cells exhibit silver grains from tritium emissions. The identity of the proliferating cells could then be determined by simultaneous visualization with antibodies specific for various cell types. UCHL1+ (CD45RO+) T cells (recall antigen-reactive helper T-cell subset) constituted 36.6 +/- 3.1% (mean +/- SEM, n = 6) of the proliferating dermal cells in involved skin, whereas Leu 18+ (CD45RA+) T cells (recall antigen naive T-cell subsets) comprised only 8.7 +/- 1.5% (n = 6). The Factor XIIIa+ dermal perivascular dendritic cell subset (24.9 +/- 1.5% of proliferating dermal cells, n = 6) and Factor VIII+ endothelial cells (23.0 +/- 2.3%, n = 6) represented the two other major proliferating populations in lesional psoriatic dermis. Differentiated tissue macrophages, identified by phase microscopy as melanophages or by immunostaining with antibodies to Leu M1 (CD15) or myeloid histiocyte antigen, comprised less than 5% of the proliferating population in either skin type. In addition to calculating the relative proportions of these cells to each other as percent, we also determined the density of cells, in cells/mm2 of tissue. The density of proliferating cells within these populations was increased in involved versus uninvolved skin: UCHL1+, 9.0 +/- 1.7 cells/mm2 versus 1.8 +/- 0.6 cells/mm2, p less than 0.01; Factor XIIIa+, 6.0 +/- 0.7 cells/mm2 versus 1.5 +/- 0.5 cells/mm2, p less than 0.01; Factor VIII+, 5.5 +/- 1.4 cells/mm2 versus 0.0 cells/mm2, p less than 0.05. The presence of preferential active proliferation of a T-cell subset in lesional dermis suggests that activating signals specific for this subset are contained within the psoriatic dermis in vivo. The activation of recall antigen-reactive T cells may be a driving force behind the dendritic cell and endothelial cell proliferation. Alternatively, the selective proliferation and expansion of these two constitutive cell types (Factor XIIIa+ and Factor VIII+) may result in signals that promote activation of UCHL1+ (CD45RO+) T cells.

Percutaneous penetration of methylglyoxal bis(guanylhydrazone): effects on hairless mouse epidermis in vivo.

Topical methylglyoxal bis(guanylhydrazone) (MGBG) previously has been shown to produce partial clinical improvement in psoriasis. To enhance therapeutic activity, studies were undertaken to optimize MGBG percutaneous penetration in vitro and to study biochemical changes related to epidermal proliferation in vivo. MGBG penetration in saline, Vehicle N, decylmethylsulfoxide, and N-methylpyrrolidone was determined in normal human skin in vitro. Maximum penetration was obtained with 10% MGBG in Vehicle N (3 micrograms/h/cm2). Both topical and systemic MGBG resulted in increased levels of S-adenosyl-L-methionine decarboxylase, suggesting an extended half-life as a consequence of MGBG binding. Topical treatment with 10% MGBG in Vehicle N also resulted in decreased epidermal polyamine levels. The changes in polyamine metabolism were also associated with inhibition of epidermal DNA synthesis. These studies suggest that this topical MGBG formulation may be a candidate for use in the treatment of psoriasis and other hyperproliferative cutaneous diseases associated with increased polyamine synthesis.

Ro 20-1724: an agent that significantly improves psoriatic lesions in double-blind clinical trials.

Two double-blind studies comparing the effectiveness of the cyclic nucleotide-altering agent (4-[3-butoxy-4-methoxybenzyl]-2-imidazolidinone) (Ro 20-1724) vs vehicle have demonstrated that this compound can improve psoriatic lesions. Although Ro 20-1724 was not as effective as intensive occlusive treatment of psoriatic lesions with 0.025% triamcinolone acetonide, Ro 20-1724 had no adverse systemic or cutaneous effects. Ro 20-1724 and other cyclic nucleotide-altering agents may have therapeutic potential in the future treatment of psoriasis.

Factors affecting human percutaneous penetration of methotrexate and its analogues in vitro.

Factors affecting the percutaneous penetration of methotrexate (MTX) and its analogues through excised human skin were studied. With application of 0.05% MTX solution to the epidermis, 0.07% of the applied dose was recovered in the epidermis and less than 0.005% penetrated completely through the skin. The percent of drug which penetrated remained constant from 0.05% to 2.0% MTX and was also unchanged by increasing incubation temperature from 28 degrees C to 37 degrees C. Cellophane tape stripping of stratum corneum following drug incubation removed essentially all the MTX that had been measured in whole epidermis. Binding of MTX was localized to the outermost layers of the stratum corneum. With removal of the stratum corneum prior to drug incubation, 25% of the applied MTX penetrated through the skin. Various vehicles were tested to determine their effect on MTX percutaneous absorption. Dimethylsulfoxide (80%), dimethylacetamide (25%), or retinoic acid (0.1%) had no effect on penetration. Maximum enhancement of penetration by 25-fold and 143-fold was obtained with retinoic acid (saturated solution in aqueous ethanol) and C-10-methylsulfoxide (2.5%), respectively. The penetration of lipid-soluble derivatives of MTX was also tested. The compound that penetrated best was the dimethyl ester of dichloro-MTX with a 3-fold increase in penetration over MTX.

Characterization of cutaneous phototoxicity induced by topical alpha-terthienyl and ultraviolet A radiation.

Alpha-terthienyl (alpha-T), a phototoxic thiophene compound isolated from marigolds (Tagetes species), affects cell membranes and does not appear to induce cytogenetic damage. This study was undertaken to investigate topical delivery of alpha-T and characterize its cutaneous phototoxicity in combination with long-wave UV radiation (UVA) in comparison with locally (intradermal) administered alpha-T. Percutaneous penetration (PC) of 0.1% and 1% alpha-T in a 3% Azone gel vehicle was studied in guinea pig skin in vitro and quantitated by UV fluorescence microscopy. Dose-dependent PC of epidermis, adnexae, and superficial dermis was demonstrated in vitro. Alpha-terthienyl (0.1% and 1%) in this vehicle was applied topically in vivo and irradiated with 30 J/cm2 UVA at intervals of 10 min-24 h. Maximum sensitization was achieved with irradiation 1 h following drug application. The clinical response was dose-dependent consisting of erythema, edema, crusting, erosion, and inhibition of hair growth and was observed 72 h to 7 days postirradiation. A comparable dose-dependent phototoxic response was observed when 5-500 micrograms alpha-T were injected intradermally and irradiated with UVA. These results indicated that low-dose topical alpha-T in a nonirritating vehicle can rapidly produce cutaneous photosensitization. Topical alpha-T/UVA may provide a selective and safer alternative approach for the photochemotherapy of psoriasis and other cutaneous diseases.