Cell cycle analysis by flow cytometry of non-exposed, sun-exposed, and tretinointreated skin I h e |jereentage of keratinoeytes in the j^rohferative jjhase of the eell cycle (S -I- G^ + M) was measured by DNA flow cytometry in sun-e.xposed, ncjn-exposed, and tretinoin-treated skin. Before tretinoiii (realment, (he pereentage of keratinoeytes actively cycling was higher m sun-exposed than in non-suii-exposed skin (p = .002) and was correlated with elinically assessed photodamage {p — .007). Subsequently, (re(inoin-treated sun-exposed skin was conipared to the pre-treatment sun-exposed skin. Overall, there was no statistically significant change. However, there was a (rend toward a deerease in the pereentage of keratinoeytes in the S -f- G2 + M phases immediately after four months of tretinoiii use that was limited to the most severely damaged patients. This effect was no longer evident two months after diseontinuing treatment. This is the first study, to our knowledge, utilizing flow eytometry to investigate the effeets of tretinoin in i)atieius with varying degress of photodamage. Nedorost ST, Bergfeld VVF, Valeir/.tiela R, Guitart J, Fleming MG. Cell cycle analysis by flow cytcjnietry of non-exposed, sunexposed, and tretinoin-treated skin. J Cutan Pathol 1992: 19: 371-375.

Re(inoie acid is a metabolite of vitamin A that affeets cellular difreren(ia(i()n Ijy modulating the ex]5ression of gene products (I). The drug has been used in hy|jerkera(otic and neoplastic conditions (2) and in androgenetie alopeeia (3). For cutaneous ne()])lasias, both prophylactic (4) and therapeutic (5) benefits have been shown. Topically applied all-trans-retinoic acid (tretinoin) is not cytotoxic to neoplastic cells, but it may increase penetration of cytostatic agents such as 5-fluorouraeil when used in combination (6). Rather, preneoplastic cells ajjpear to be steered toward normal dillerentiation. Topieal tretinoin reverses actinic damage as assessed by routine histolcjgy. Histologie changes resulting from tretinoin treatment of photo-damaged skin include: 1) elimination of keratinoeyte dysplasia, 2) normalized orientation of epidermal layers, 3) elimination of clumping of melanin granules in basal eells, 4) correction of atrophy ofsubcorneal cell layers, and 5) decreased hy]3erkeratosis (7). These histologic

Susan T. Nedorost, Wilma F. Bergfeld, Rafael Valenzuela, Joan Guitart, Matthew G. Fleming Departments of Dermatology, and Division of Laboratory Medicine, Cleveland Clinic Foundation, Cleveland. Ohio, USA.

Susan T. Nedorost, Associates in Dermatology Inc., 14701 Detroit Ave. Lakewood, OH 44107, USA. Accepted April 2, 1992

changes suggest inereased replieation rate of keratinoeytes with earlier shedding from the stratum eornetim (7). This is supported by the finding that in some patients tretinoin inereases (he pereentage of cells in the synthetic phases of the eell cycle as measured by cyto]:>hotometry. This is probably done by reeruiting noneyeling eells into the synthetic or cycling phase (7). Analysis of cellular DNA by flow cytometry gives information on the percentage ol' eells in the different stages of the cell eycle and on ploidy (8). It is faster and more accurate than prex'ious photometric methods (8). DNA measurements are obtained by staining the nuelei with a DN.A-binding fluoroehrome whieh is excited by a laser beam. As single nuclei are illtiminated by a focused laser beam, the lluoreseence intensity emitted by the nuelei whieh is proportional to their DNA content is measured and then subjecled lo eomputerized analysis to produce a DNA histogram (Fig. 2) (9). For example, 2e DNA content as measured by fluorescenee intensity

371

Nedorost et al. l''ig. I. (Jaling leelmicine used to sc|)arate single nuclei from clumped nuclei and debris belc:>re analysis of DNA histogram, fwcnts outside the set elcc tronic windc5W arc exchidcd Irom tDNA analysis.

2iaQ

B B

T—I—I—I—!—I—r BBB

L2

BBS

t BOB

\i

corresponds to cells in phase GQ—G,; cells with 2-4c DNA content are in S (synthetic) phase; and 4e DNA eontent eorresponds to cells in G^ + M phase. We examined sun-exposed, non-exposed, and tretinoin-treated skin by flow cytometry to look for effeets of ehronie sun exposure and tretinoin treatment on (he cell cycle.

Material and methods

Four mm |3uiich biopsies of skin were obtained Irom 16 moderately-to-severely sun-damaged patients (14 women and 2 men, ages 35-69 years). The most severely damaged patients had a history of aednie keratoses and/or basal cell eareinomas. Thirteen patients completed the study. Four biopsies were taken from each of these 13 patients: from abclomi-

tn

a c 3

372

/'Vi,'. 2. Example of DNA histogram. Large peak on lelt is an inlernal standard (chic ken red blood cell luu.lei). Middle peak is (;„-(;, (channel 1:53). Small peak al rigln is G., + M (channel 2(i()). Cell cycle statistics in this case: G,,-G| = 93.ii"/«, S2.2%, G.,-M = :^.9%. The CV of the (;,,-(5| peak = 2.97n.

Cell cycle analysis Table 1. Overall comparison of sun-exposed and non-exposed sifes

Table 3. Correlation analysis between clinical severity of photodamage

Baseline

Forearm Abdomen

N

Mean % cycling

STD

15 16

4.09 2.57

1.33 .61

Forearm Abdomen

N

Pearson correlation coefficient

p-value

15 16

.663 -.224

.007 .405

p-value - .002

nal (non-sun-exposed) skin and from forearm (stiiiexposed) skin at baseline, after four months of treatment, and at one to two months after diseontinuatiori of tretinoin. Treatment consisted of application of tretinoin .05% cream to the forearms every night. Biopsies were taken from the proximal portion of the distal third of the dorsal forearm. Subsequent biopsies were taken at least 2 cm from previous biopsy sites. Obvicius lentigines and actinic keratoses were avoided. All biopsies were obtained in the late morning or early afternoon. None of the patients had had a recent signifkant sun-exposure. Patients were assessed for clinical severity of photodamage and ranked from 2 (mild-to- moderate) to 5 (severe) based on texture, eolor (including lenligines), and degree of mottled i^igmentation. The amount of tretinoiii eream used over the four-month treatment period was delermined by weighing the tubes. The skin specimens were incubated at 4°C in .5% aeetic acid for 48-72 h to facilitate blunt disseetion of the epidermis. (10) Histologic sections of the remaining dermis were examined to eonfirm eomplete epidermal removal. The epidermis was then plaeed in D M S O citrate bufler, snap frozen in liquid nitrogen, and stored at — 70°C fbr one to five weeks. (11) After thawing, the epidermis was incubated in a solution containing Nomidet-P40, trypsin, and spermine tetrahydrochloride at pH 7.6 for 70 min at room temperature to liberate the keratinoeyte nuclei. This step was followed by a 10-min incubation in a solution containing trypsin inhibitor and ribonuelease A. Finally, the nuclei were stained with propidium iodide and spermine tetrahydrochloride at pH 7.6 as previously described. (11) The nuclei were then filtered through a 30 micrometer nylon mesh.

Flow eytometric analysis of the nuelear DNA content was performed with a Beeton-Dickinson FACSAN Mow cytometer. Nuelei were gated using 2parameter (pulse area versus pulse width) displays to eliminate duplex and other cell aggregates (Fig. 1). The DNA histogram was analyzed with the R F I T software program (Fig. 2). Chicken red blood eell nuelei were used as an internal standard, and normal human peripheral blood lymphocytes as a diploid eell standard. A previous study of flow eytometry using normal epidermis prepared wilh methods similar to ours demonstrated that small amounts of dermal eontamination and variation in frozen storage time up to one month did not signifieantly alter results. (9) Diurnal variation has a greater effect on the 0., -I- M than the S fraction. (12) A paired t-test was used to assess the diflerenee in percentage of keratinoeytes in the S -1- Go + M phases between abdominal and forearm skin. Correlation analysis (Pearson) was used to assess the relationship between percentage of eells in the S -1- G > -f M phases in forearm and in abdominal sites and clinical degree of photodamage. Analysis of variance was used to compare the percentage of keratinoeytes in the S -f- G^, -I- M phases in forearm skin at baseline to the pereentage in the S H- G > -h M phases after four months of treatment and two months after diseontinuing treatment in patients with different severities of photodamage. Beeause of small sample size, patients in the two groups with the least photodamage (Grou|3S 2 and 3) were eombined for this analysis and eompared to the most severely damaged groups.

Table 2. Comparison between sun-exposed and non-exposed sites by degree of photodamage Abdominal baseline

Forearm baseline Severity 2 3 4 5

N

Mean % cycling

STD

1 4 7 3

3.3 3.25 3.86 6.00

0.38 1.25 0.36

Severity 2 3 4 5

N

Mean % cycling

1 4 8 3

2.9 2.4 2.82 2.00

STD

0.60

0.47 0.62

373

Nedorost et al. Table 4. Change in % cycling immediately after four months use compared to baseline* Severity 2 3 4 5

N

Mean change

STD

1 3

-0.50 2.50 0.25 -1.17

1.39 0.95 0.21

4 3

p-value = .052 'Groups 2 and 3 combined for analysis

Results The percentage of keratinoeytes in the S -I- Gj + M phases in baseline forearm (sun-exposed) skin was greater than in the abdominal (non-sun-exposed) skin (p = 0.002) (Table 1). Patients with a greater degree of photodamage by elinieal assessment had a larger pereentage of eells in the S -I- G2 + M phases in forearm skin eompared to less damaged patients. There was no eorrelation between elinieal degree of photodamage in the forearm and pereentage of eells in the S -f- G2 + M phases in the abdominal (non-sun-exposed) skin (Tables 2 and 3). There was a decrease in the percentage of cells in the S -f G2 + M phases immediately after treatment in the most severely ]3hotcj-damaged patients (p = .052) (Table 4). This trend was not evident one to two months after diseontinuing tretinoin (p = .856) (Table 5). However, overall there was no signifieant change in the pereentage of cell in the S -|- G^ + M phases in forearm skin either immediately after four months of tretinoin use or one to two months after diseontinuation of tretinoin (p-values = .438 and .795, respectively). There was also no signifieant correlation between the amount of tretinoin used and the change in percentage of cells in the S -f Gj + M phases at these intervals. The mean amount of tretinoin used over the four months of treatment to forearms was 45.5 gm ± 19.8 gm. The; amount of tretinoin used by each group of patients is shown in Table 6. Interestingly, patients who had a higher degree of clinically assessed tretinoin-induced irritation after two months of treatment used signifieantly less tretinoin compared to less irritated patients (p = .O5). There was no significant correlation fjetween degree of tretinoin-induced irritation after two and four months of treatment and the change in pereentage of cells in the S + G2 + M phases.

Discussion Regional variation in the percentage of eells in S phase has been demonstrated previously by flow cytometry. Forearm skin was shown to have more

374

Table 5. Change in % cycling 1-2 months after discontinuing fretinoin compared to baseline' Severity 2 3 4 5

N

Mean change

STD

1 3

0.80 -0.63 0.20 -0.40

1.80 1.47 0.00

4 2

p-value = .856 * Groups 2 and 3 combined for analysis

eells in S phase than in thigh or lower abdominal skin. This regional variation was much greater than variation between patients of diflerent age or sex. (13) Previously, non-sun-exposed skin studied by use of a dye marker to measure stratum eorneum renewal time was shown to have prolonged epidermal renewal time in patients over 50 years of age. (14) In our study, the most severely sun-damaged patients, who tended to be older, did have slightly fewer cells from non-sun-exposed skin in the S -f- G2 + M phases than the generally younger, less severely damaged patients. However, this weak trend based on age was greatly outweighed by the inerease in eells in the S -I- G^ + M phases in the sun-exposed skin. Thus, the effeets of photoaging on eell proliferation are far greater than the efTects of intrinsic aging. Our study shows that the elinieal assessment of severity of photodamage correlates with the percentage of cells in the proliferative phase of the cell cycle. A change in this pereentage reflects either a change in cell cycle length or a change in the number of resting eells in the eell population. Previous studies have shown that there is an increase in labeling index (15) and of proliferating eells as measured by flow cytometry (16) in epidermal tumor eells compared to perilesional skin, and in perilesional skin eompared to unexposed skin. Clinically normal skin of patients with basal cell eareinoma or Bowen's disease has also been shown by flow eytometry to have more cells in S phase than eontrol skin. (17) The trend toward a decrease in the pereentage of eells in the S -f Gj -f- M phases after tretinoin use in the most severely photodamaged patients may suggest that tretinoin reverses the hyperprolileration of Table 6. Amount of tretinoin used in each group by degree of photo damage Amount of tretinoin used: (grams) Severity

N

Mean

STD

2 3 4 5

1 3 6 3

56.00 43.27 44.95 45.47

33.23 14.83 25.14

Cell cycle analysis actinically damaged skin. This may im|:)ly regression of premalignant ehanges. A previous study doeumenting eosmetie imjirovement after 16 weeks of tretinoin therapy reported an increased number of mitoses in tretinoin-treated compared to placebo-treated skin. (18) This explains the eorreetion of aetinie atrophy attributed to tretinoin, (7) but it is difRcult to reconcile with our findings of decreased epidermal proliferation in severely damaged patients. This diserepancy may be due to the inclusion of more severely damaged patients in our study. Our less severely damaged patients also showed an inerease in eell ]:)roliferation which refleels reeiuilment of noneyeling cells into the proliferating pool or augmentation of repair processes. We pro])ose ihat the opposite efleet (decreasing cell prcjliferatioii) noted in our most severely damaged jjatients may be due to normalization of premalignant cells which had reached a hyperproliferative state. Complete epidermal turnover may explain the return to a high percentage of eyeling eells onee (he tieatmenl has been diseontinued for two months. 1 his is the first study, [o our knowledge, utilizing" flow eytometry to investigate the eflects of tretinoin in patients with varying degress of photodamage. Our hypothesis is based on a small number of patients. Further sttidy is neeessary to confirm our theory. Acknowledgements We wish to thank Lata Paranandi, MSHP and Sharon Vanderbrug Medendorp, MPFl fbr their assistance with the statistical analysis. Also, thanks to Bettie Haugabrook, PCA and Linda Langfbid for their assistanee with the elinieal aspeets of the study, to Rebecca Turinie, BA, ASCP, IS for her skillful technical assistance, and to Carol White fbr typing the manuscript. Partial funding fbr this study was prcnided by Ortho Pharmaceuticals, Inc.

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