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University Microfilms International 300 N. Zeeb Road Ann Arbor, Ml 48106

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Albrightson, Christine Ruth

THE EFFECT OF 8-METHOXYPSORALEN ON THE CYCLIC-AMP CONCENTRATION OF NORMAL HUMAN FIBROBLASTS IN VITRO

The Ohio State University Ph.D. 1983

University Microfilms International 300 N. Zeeb Road, Ann Arbor, MI 48105

THE EFFECT OF 8-METHOXYPSORALEN OR THE CYCLIC AMP

CONCENTRATION OF NORMAL HUMAN FIBROBLASTS IN VITRO

......

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree of Doctor of Philosophy in the Graduate

School of The Ohio State University

Christine Ruth Albrightson

*****

The Ohio State University

1983

Reading Committee: Approved By:

R. Fertel J. Bianchine S. Tjioe R i o W X F e v Ui^ R. Stephens______:______Advisor Department of Pharmacology ACKNOWLEGEMENTS

I would like to thank my advisor. Dr. Richard Fertel. for his guidance, understanding, encouragement and friendship-

I will strive to extend that which he has helped me to achieve.

I wish to thank my reading committee, Drs. J. Bianchine,

R. Stephens and S- Tjioe for their critical evaluation of my thesis.

I would like to acknowledge the OSU Cell Culture Service and the friends who work there; Lori, Laura, Kelly, Pat,

Denny, Bill and Deb. I would also like to acknowledge the

OSU Tumor Procurement and Pathology Laboratory-

The laboratory assistance from Cathy, Pam and Barb was greatly appreciated and their friendship will always be cherished.

Thanks are also extended to my fellow graduate students;

Sharon, Cindy, Dennis, Roy, Mike, George, Pam and Charlie for their assistance, discussions and friendship.

I am especially grateful to my parents and sister for their love, support and encouragement. VITA

NAME: CHRISTINE R- ALBRIGHTSON

EDUCATION: 1979 - present Graduate Research Assoc. The Ohio State University, Department of Pharmacology Columbus. Ohio

1978 - 1979 The Ohio State University Continuing Education

1971 - 1974 The Ohio State University B.S., Biology

1970 - 1971 University of Rhode Island Kingston, R.I.

HONORS

The OSU Graduate Student Alumni Research Award, 1981.

The Jack Van Fossen Award for the outstanding graduate student in Pharmacology, 1982.

ICSABER Award for outstanding research, 1982.

The OSU Presidential Fellowship, 1982-1983-

PUBLICATIONS

Abstracts

Hall KY, Albrightson CR and Hart RW: A direct relationship among primates between maximum life span and DNA repair.

i l i Proceedings of the International Meeting of Gerontological Society. Tokyo, Japan. 197 8.

Hart RW, Hall KY. Albrightson CR and Sacher GA: Evaluation of longevity and DNA repair processes in mammals. Proceedings of the International Meeting of the Gerontological Society, Tokyo, Japan. 1978.

Yates AJ, Thompson DK, Boesel CP- Hart RW and Albrightson CR: Lipid composition of human gliomas- Transaction of American Society for Neural Chemistry, 1978.

Fertel RH, Albrightson CR and Hart RW: The effect of tetradeconoyl phorbol acetate (TPA) in cyclic nucleotide concentration in human fibroblasts- OLACC- 1979.

Albrightson CR and Fertel RH: Extranuclear effects with 8-methoxypsoralen and ultraviolet irradiation- ICSABER, 1980

Albrightson CR- Fertel RH, Stephens R and Brown BV: Extranuclear effects of 8-methoxypsoralen (8-MOP) on normal human fibroblasts in vitro. Federation Proceedings, 40:730, 1981.

Albrightson CR and Fertel: Psoralens alter the cyclic AMP of human cells. ASPET/SOT Meeting, the Pharmacologist 24(3):157 . 1982.

Journal articles

Yates AJ, Thompson DK, Boesel CP, Albrightson CR and Hart RW: Lipid composition of human neural tumors. J Lipid Res 20:428-436, 1979.

Fertel RH, Tejwani GA, Albrightson CR and Hart RW: The effect of ultraviolet light on the cyclic nucleotide system of human fibroblasts- Photochem Photobiol 34:275-278, 1981.

Albrightson CR. Fertel RH, Stephens R and Brown BV: Psoralens increase the concentration of cyclic AMP in normal human fibroblasts and monocytes in yitjo. Submitted. TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ii

VITA iii

LIST OF TABLES viii

LIST OF FIGURES ix

LIST OF PLATES xiii

INTRODUCTION 1

A. Manifestation and etiology of psoriasis 1

B. Treatment of psoriasis 3

C. The use of the psoralens in psoriasis 4

D. The role of cyclic nucleotides in the regulation of psoriatic epithelium 6

E. Overview of experiments 7

METHODS 10

A. Tissue culture materials 10

1. Culture media supplements 10

2. Cell culture media 10

3. Balanced salt solutions 11

4. Enzyme solutions 11

B. Cell culture techniques 11

1. Establishment of fibroblast cultures 11

2. Establishment of epithelial cultures 15 3. Collagen coating technique 16

4. Method for counting cells 17

5. Cryostorage of cells in culture 18

C. Preparation and treatment of cells in culture for the cyclic nucleotide assays 20

1. Preparation of fibroblast cultures 20

2. Preparation of epithelial cultures 20

3. UV-C irradiation 21

4. Treatment of fibroblast and epithelial cultures 21

5. Preparation and treatment of monocytes 22

6. Materials 22

D. Preparation of fibroblast cultures for enzyme analysis 22

1. Preparation of fibroblast cultures 23

2. Preparation of enzymes 23

E. Cyclic nucleotide radioimmunoassay 24

F. Protein assay 25

G. Adenylyl cyclase assay 26

H . Cyclic AMP phosphodiesterase assay 28

I. Prostaglandin radioimmunoassay 32

J. Cytofluorometry of human cells 33

K. Statistical analysis 35

RESULTS 36

A. The effect of ultraviolet light on cyclic AMP 36

B. The effect of UV light and PABA on cyclic AMP 42

C. The effect of UV light and 8-MOP on cyclic AMP 45

vi D. The effect of 8-MOP on cyclic AMP 48

E. The long term effect of 8-MOP treatment on fibroblasts 53

F. The effect of 8-MOP on the cyclic AMP of epithelial cells and monocytes 64

G. The effect of psoralen analogs on cyclic AMP 79

H. The effect of adenylyl cyclase receptor blockers on the 8-MOP mediated increase in cyclic AMP 87

I. The effect of 8-MOP on prostaglandins 97

J. The effect of 8-MOP on adenylyl cyclase 98

K. The effect of 8-MOP on phosphodiesterase 107

DISCUSSION 117

A. The biological effects of ultraviolet light 117

B. Effects of psoralens 123

C. Effects of 8-MOP on other cell types 128

D. Effect of psoralen analogs on cyclic AMP 129

E. Mechanism of 8-MOP action on cyclic AMP 130

D. Significance 137

BIBLIOGRAPHY 141 LIST OF TABLES

Table Page

1 The effect of adenylyl cyclase agonists and antagonists on the concentration of cyclic AMP in human fibroblasts 89

2 The effect of adenylyl cyclase receptor blockers on the rise in cyclic AMP induced by 8-MOP 91

3 The effect of pretreatment with psoralen analogs on the ability of 8-MOP to increase cyclic AMP in fibroblasts 94

4 The effect of pretreatment with hydrogenated psoralen analogs on the ability of 8-MOP to increase cyclic AMP in fibroblasts 96 LIST OF FIGURES

Figure Page

1 The effect of time in culture on the cyclic AMP concentration of normal human fibroblasts 37

2 The effect of time in culture on the cyclic GMP concentration of normal human fibroblasts 38

3 The effect of ultraviolet irradiation on the cyclic AMP concentration of human fibroblasts 44 hours after plating 40

4 The effect of ultraviolet irradiation on the cyclic AMP concentration of human fibroblasts 96 hours after plating 41

5 The dose dependent effect of UV irradiation on the cyclic AMP concentration of fibroblasts 43

6 The effect of PABA on the UV-induced rise in cyclic AMP 44

7 The effect of 8-MOP and UV irradiation on the cyclic AMP concentration of fibroblasts 46

8 The time dependent effect of 8-MOP on the cyclic AMP concentration of fibroblasts 49

9 The dose dependent effect of 8-MOP on the cyclic AMP concentration of fibroblasts 50

10 The time dependent effect of 8-MOP on the cyclic AMP concentration of fibroblasts when treated under red light 51

11 The dose dependent effect of 8-MOP on the cyclic AMP concentration of fibroblasts when treated under red light 52

12 The time dependent effect of 8-MOP on the cyclic GMP concentration of fibroblasts when treated under red light 54

ix 13 The dose dependent effect of 8-MOP on the cyclic GMP concentration of fibroblasts when treated under red light 55

14 The effect of 8-MOP treatment on fibroblast cultures at different times after plating 56

15 The effect of extended incubation with 8-MOP on the cyclic AMP concentration of fibroblasts 58

16 The effect of extended incubation with 8-MOP on the cyclic GMP concentration of fibroblasts 59

17 The effect of extended incubation with 8-MOP on the ratio of cyclic AMP to cyclic GMP 60

18 The effect of extended incubation with 8-MOP on fibroblast cell number 62

19 The effect of extended incubation with 8-MOP on the protein content of fibroblast cultures 63

20 The DNA fluorescence pattern 12 hours after 8-MOP treatment compared to controls 66

21 The DNA fluorescence pattern 36 hours after 8-MOP treatment compared to controls 68

22 The time dependent effect of 8-MOP on the cyclic AMP concentration of human epithelial cells in culture 70

23 The dose dependent effect of 8-MOP on the cyclic AMP concentration of human epithelial cells in culture 71

24 The time dependent effect of 8-MOP on the cyclic GMP concentration of human epithelial cells in culture 72

25 The dose dependent effect of 8-MOP on the cyclic GMP concentration of human epithelial cells in culture 73

26 The effect of time in culture on the cyclic AMP concentration of human epithelial cells 74

27 The effect of time in culture on the cyclic GMP concentration of human epithelial cells 75

28 The effect of time in culture on the protein

X content of human epithelial cells 77

29 The effect of 8-MOP on the concentration of cyclic AMP in human peripheral blood monocytes 7 8

30 The time dependent effect of TMP on the cyclic AMP concentration of human fibroblasts in culture 80

31 The dose dependent effect of TMP on the cyclic AMP concentration of fibroblasts in culture 81

32 The effect of psoralen analogs on the cyclic AMP concentration of fibroblasts in culture 84

33 The effect of 8-MOP hydrogenated analogs on the cyclic AMP concentration of fibroblasts 86

34 The time dependent effect of 8-MOP on the production of PGE2 by fibroblasts in culture 99

35 The time dependent effect of 8-MOP on the production of PGF2o< by fibroblasts in culture 100

36 The time dependent effect of 8-MOP on the production of prostacyclin by fibroblasts in culture 101

37 The time dependent effect of 8-MOP on the production of thromboxane A2 by fibroblasts in culture 102

38 The effect of 8-MOP and known AC activators on the activity of adenylyl cyclase obtained from fibroblast cultures 104

39 The effect of AC activators in combination with isoproterenol or 8-MOP on the activity of adenylyl cyclase from fibroblast cultures 106

40 The time dependent effect of isoproterenol on the cyclic AMP concentration of fibroblasts in culture 108

41 The dose dependent effect of isoproterenol on the cyclic AMP concentration of fibroblasts in culture 109

42 The time dependent effect of papaverine on the cyclic AMP concentration of fibroblasts in culture 110

xi 43 The dose dependent effect of papaverine on the cyclic AMP concentration of fibroblasts in culture 111

44 The time dependent effect of isobutylmethylxanthine on the cyclic AMP concentration of fibroblasts in culture 112

45 The dose dependent effect of isobutylmethylxanthine on the cyclic AMP concentration of fibroblasts in culture 113

46 The km of cyclic AMP phosphodiesterase of human fibroblasts 115

47 The of 8-MOP for the cyclic AMP phophodiesterase of human fibroblasts 116 LIST OP PLATES

The molecular structure of 8-methoxypsoralen and trimethylpsoralen

The molecular structure of 5-methoxypsoralen. 3-carbethoxypsoralen and 5-methylisopsoraien

The molecular structure of monhydrogenated and dihydrogenated 8-methoxypsoralen INTRODUCTION

MANIFESTATION AND ETIOLOGY OF PSORIASIS

Psoriasis is a major skin disease of unknown etiology which affects 1-3 million persons in the United States (1).

Clinically, it is manifested by inflamed and thickened epithelium with sharp margination and characteristic scale.

These stigmatizing chronic recurrent lesions are often physically and emotionally debilitating to the patient (1).

There is no cure for psoriasis, and treatment often encompasses a lifetime.

Histologically, psoriasis is characterized by abnormal epithelial cell proliferation and maturation, resulting in a high number of basal cell mitoses, abnormal keratinization, and absence of the stratum granulosum (2). Dermal hyperplasia coincides with enlarged dermal papillae (2) and is associated with morphologically and functionally abnormal capillary loops (2,3) . There is also an increase in the number and type of immune competent cells present in both the epithelium and dermis.

Psoriasis is a disease with a tendency toward heritability (4,5). Abnormal humoral substances as well as immune cell and epithelial cell dysfunctions have been suggested as causative factors in psoriasis. The dermis is essential in the maintenance of epithelial proliferation,

but its role in the primary etiology of the disease is

unknown. While environmental conditions may modify the

course, age of onset and severity of the disease, little is

known of the circumstances which cause a spontaneous

eruption of a proliferative lesion in previously unaffected

epidermis.

Unfortunately, research into the pathogenesis of

psoriasis has been hampered by the lack of a reliable animal

model. Most of the information about the pathogenesis of

this process has been obtained from the careful study of

cells from human skin lesions, by means of direct

microscopic examination as well as diverse cell and organ

culture techniques.

Kinetic in vivo studies of human skin indicated that

psoriatic epidermal cells turn over more rapidly then

uninvolved or normal skin (6). This high turnover rate

results in the abnormal keratinization and

hyperproliferation seen in psoriatic epidermis. The general

increase in turnover rate could be due to an increased

germinative cell population (7), a pathologic cell cycle

with a decrease in DNA synthesis time (S phase) (6) or to a

recruitment of cells that are normally blocked at or

G 2 (8,9,10). A defect may reside in either the factors which determine the length of the cell cycle or the factors

which determine how many and which cells are to proliferate. 3

TREATMENT OF PSORIASIS

Numerous topical and systemic drugs have been used in the treatment of psoriasis with less than satisfactory results.

Topical therapy has been the mainstay in the treatment of psoriasis. The major drawbacks of topical therapy are patient compliance and uncertainty about the percutaneous absorption of the drug. Among the many topical agents used, glucocorticosteroids are the most effective. However, they are less than curative. Unfortunately, more potent topical steroids have a greater local or systemic toxicity (1). A second class of topical agents includes crude coal tar, which has been used by itself and in combination with ultraviolet light (Goeckerman Regime) (11) . Anthralin or dithranol have also been used alone (12) and in combination with coal tar and ultraviolet irradiation (1). Among the systemic agents, the antimetabolites are the most effective.

However, when given systemically, they produce hematological suppression, liver abnormalities, gastrointestinal irritation and bleeding, teratogenic effects and mutagenic changes (13).

Other systemic agents include retinoids and steroids used alone, and 8-methoxypsoralen (8-MOP) used in combination with ultraviolet light (PUVA). The retinoids affect the growth and differentiation of epithelial cells, but their usefulness is limited due to a high frequency of side effects (1). However, they are potentially beneficial when used in combination with other therapies (14,15). The systemic use of steroids carries with it a high potential for systemic toxicities. In addition, patients develop resistance to the steroids, and the psoriatic lesions commonly reoccur when steroid therapy is discontinued (13).

Although ionizing irradiation is no longer used in the treatment of psoriasis, ultraviolet irradiation, both alone and in combination with a variety of agents has proven beneficial (1) . UV irradiation in combination with

8-methoxypsoralen has proven particularly effective (16).

THE USE OF THE PSORALENS IN PSORIASIS

The furocoumarins, or psoralens, are tricyclic compounds

(plate 1) derived from the plant Ammi Hajus.

8-methoxypsoralen (8-MOP) and 4,5',8-trimethylpsoralen (TMP) were initially used in the treatment of vitiligo (17) and, more recently, 8-MOP has proven beneficial in the photochemotherapy of psoriasis (16) . The major short-term side effects of the psoralens include nausea, erythema and pruritus (18) . It has been demonstrated that psoralen binds to pyrimidine bases of DNA, and, in combination with ultraviolet light at 315-400 nm (UVA), forms photo-adducts

(19). This alteration of the DNA molecule decreases DNA synthesis (20,21) and causes cell death (22,23), both of which may result in the eventual diminution in cellular OCH_ o

8-METH0XYPS0RRLEN C8 MOP)

4,5', 8-TRIMETHYLPSORRLEN (TMP)

PLATE 1. THE MOLECULAR STRUCTURE OF 8-METHOXYPSORALEN AND

TRIMETHYLPSORALEN proliferation seen after PUVA treatment. The possible long-term problems with PUVA therapy include the development of multiple malignancies of the skin (24,25,26) and possible premature cataract formation (27).

The interaction of the psoralen molecule with DNA does not explain all of the effects of PUVA. Some effects of PUVA extend beyond the time needed for repair of the DNA damage

(28). Preliminary signs of clearing (normalization of capillary loops (29) and return of the stratum granulosum

(30)) precede a decrease in basal cell proliferation rate as determined by a decrease in the labeling and mitotic indices. In addition, reports indicate that PUVA treatment alters certain immunological functions (31,32), increases melanocyte number (33,34) and increases melanogensis

(35,36). These effects may not be completely attributable to the DNA damage previously associated with PUVA, but rather these effects may be the conseQue^ce of the 8-MOP action upon other biochemical parai .. .. o.a the cell.

THE ROLE OF CYCLIC NUCLEOTIDES IN THE REGULATION OF

PSORIATIC EPITHELIUM

The widely recognized increase in epithelial proliferation (6) and increased glycogen content (3) of psoriatic cells, coupled with the effect of cyclic AMP on cell proliferation and glycogen metabolism (37) led Voorhees to propose that there is a defect in the cyclic AMP metabolism of psoriatic skin (38) . A number of investigators have subsequently attempted to elucidate the role of cyclic nucleotides in the hyperproliferation and decreased maturation rate of psoriatic epidermal basal cells. These studies include the determination of the cyclic AMP (39,40) and cyclic GMP (41,42) content of psoriatic lesions, the in vivo production of cyclic AMP and cyclic GMP of psoriatic tissue (43) , the activity of adenylyl cyclase (44), guanylyl cyclase (45) and phosphodiesterase (46) in psoriatic tissue, and the clearing

(47) or exacerbation of lesions by drugs which alter cyclic nucleotide metabolism (48,4°,50).

Although the precise re..- 1 the cyclic nucleotides in the etiology, progression and treatment of psoriasis has not been clearly established, their involvement has been shown.

These facts, together with the unexplained effects of PUVA on the immune cells and melanocytes as well as the importance of cyclic nucleotides in the regulation and function of these cells, formed the basis for the following in vitro study of the effects of the psoralen compounds on the cyclic nucleotide metabolism of human cells.

OVERVIEW OP EXPERIMENTS

Preliminary experiments performed in this laboratory involved the determination of the effects of UVC light

(200-280 nm) on the cyclic nucleotides of normal human fibroblasts in culture. Pharmacologic agents which were known to interact with UV light were then examined for their effects on the UV mediated effects. Experiments involving the combined use of UV irradiation and 8-MOP resulted in a finding which is the basis for the remaining experiments covered in this work. 8-MOP was found to cause a change in the cyclic AMP concentration of cells in culture independent of UV light. Cell cytotoxicity, inhibition of protein accumulation and cell proliferation was measured after treatment with 8-MOP. Epithelial cells and monocytes were tested for their cyclic nucleotide response after treatment with 8-MOP. Psoralen analogs were also tested for their effects on the cyclic nucleotide concentration of fibroblasts. The pharmacologically active psoralen, TMP, was the only other psoralen compound tested which had an effect on the cyclic nucleotide concentration of cells in culture.

The mechanism by which 8-MOP caused the rise in cyclic

AMP concentration was then analyzed. Adenylyl cyclase receptor blockers were tested for their ability to block the increase in cyclic AMP mediated by 8-MOP. The inactive psoralen analogs (as related to their inability to increase cyclic AMP) were also tested as potential blockers. The ability of 8-MOP to increase cyclic AMP in an adenylyl cyclase enzyme preparation was tested. Known adenylyl cyclase activators served as the positive controls to which 9 the activity of 8-MOP was compared. Phosphodiesterase inhibition was another mechanism by which 8-MOP could cause an increase in cyclic AMP. Papaverine and isobutylmethylxanthine dose and time dependent changes in the cyclic AMP concentration in fibroblasts in vitro were compared to that of 8-MOP. Cytosolic enzyme preparations from the fibroblast cultures were used to determine the phosphodiesterase Km values. Various substrate concentrations with increasing concentrations of 8-MOP were used to determine the for 8-MOP.

This document encompasses the initial experiment in which the observation was made that 8-MOP mediated an increased cyclic AMP concentration in normal human fibroblasts in vitro through the experiments which were designed to verify the effect and discover the mechanism by which it occurred. METHODS

I. TISSUE CULTURE TECHNIQUES

CELL CULTURE MATERIALS

A. Supplements

Fetal Calf Serum (FCS)

FCS (lot # 1L039) was obtained from M.A. Bioproducts,

Walkersville, MD.

Penicillin/streptomvcin (P/S)

Penicillin and streptomycin were both obtained from Sigma

Chemical Co., St. Louis, Mo. The concentration of P/S used yielded a consentration of 100 units of penicillin G/ml medium and 100 ug streptomycin sulfate/ml medium.

Amphoter icin_B

Amphotericin B (GIBCO, Grand Island, NY) was used at a final concentration of 250 ug/ml mediur, ,

Gentamicin sulfate

Gentamicin sulfate (Sigma) was used at a final concentration of 50 ug/ml medium.

B. Cell culture media

Medium A

Medium A was composed of the following:

Minimum Essential Medium (Eagle) - formula # 78-5048 (GIBCO) which was modified by the addition of 1.5X essential amino

10 11 acids, 1.5X vitamins, 2X non-essential amino acids and approximately IX L-glutamine. To a 10 liter package of dry medium in 3 L of double-distilled water, 4.87 g/L NaCl and

1.1 g/L sodium pyruvate (Sigma) were added. The pH was adjusted to 6.8 and then 15 g/L of NaHCO^ was added. The final volume was brought to 10 L with double-distilled water

and 5% CO2 was bubbled through the medium for approximately 5 minutes prior to sterilization by filtration. FCS was added to the medium for a final concentration of 10% prior to addition of the medium to cells.

Me<3]Lum.J3

Medium B was medium A plus IX P/S, IX gentamicin and IX amphotericin B.

C. Balanced salt solutions

Hank's balanced salt_solution (HBSS)

HBSS (GIBCO) was supplemented with 2X P/S, IX gentamicin and

IX amphotericin B.

Phosphate buffer.ed_saline (PBS)

PBS, pH 7.2, consisted of the following inorganic compounds in distilled water.

NaCl 8.0 g/L KC1 0.2 g/L Na«HPO. 1.15 g/L KH^PO^ 0.2 g/L

d . Enzyme.-S-Qlutions

Lyophilized trypsin (209 units/mg) (Worthington Biochemical 1 2

Co., Freehold, NJ) was stored at 4° C. Three different

trypsin solutions were prepared. A 0.01% solution of

trypsin (10 mg/100 ml PBS) was used tosplit fibroblast

cultures. A 0.25% solution of trypsin (250 mg/100 ml PBS)

was used to digest the epidermal-dermal junction of

foreskins. A 0.1% solution of trypsin (100 mg/100 ml PBS)

with 0.02 % EDTA (20 mg/100 ml PBS) was used to split

epithelial cell cultures. Stock solutions were stored at 4°

C for less than 2 months. Longer storage times required a

-4° C temperature.

Type II collagenase (204 units/mg) was obtained from

Worthington Biochemical Co. A 0.4% collagenase solution

(400 mg/100 ml PBS) was used to dissociate fibroblasts from

dermal tissue.

CELL CULTURE METHODS

A. Establishment _of fibroblast_cultures

Two methods (A and B) were employed to establish fibroblast

cultures. Each of these methods is outlined below:

Method_A

Method A was used for the initial establishment of primary

fibroblast cultures. Normal human foreskins were stored in

HBBS with IX penicillin/streptomycin, IX gentamycin and IX

fungizone at 4° C until processed. After storage, the

tissue was rinsed twice in phosphate buffered saline (PBS) and placed in a plastic culture dish (Corning Glass Works, Corning, NY) with 0.5 ml of 0.01% trypsin. The tissue was 3 sliced between two No. 22 scalpel blades into 1 mm

pieces, transferred into a flask containing 1 0 ml of 0 .0 1 % trypsin and stirred for 30 minutes at 37° C. The digested material was then transferred to a 15 ml centrifuge tube and centrifuged at 90 X g (Sorval centrifuge, model GLC-1) for

10 minutes. The supernatant fraction was discarded and the precipitate, which included single cells and tissue fragments from one to four foreskins, was placed into a T-75 tissue culture flask with 10 ml of medium B. Cultures were

grown at 37° C using a 5% C02 atmosphere in a humidified incubator (Forma Scientific, Marietta, OH). Viable cells and tissue explants attached to the plastic after 24 hours, at which time the medium was replaced with 15 ml of fresh medium A. Thereafter, the medium was changed every 2-3 days. The fibroblasts grew from the explants and divided until the entire surface was covered (this was termed a confluent culture). Primary cultures were designated as passage level zero (P-0). After a culture reached confluency, the medium was removed, the cells were washed twice with PBS and 3 ml of 0.01% trypsin was added to the culture. After a 10 minute incubation with trypsin at

37° C, the cells detached from the flask surface and became spherical. Detached cells were then aliquoted into 1 or 2

T-150 flasks containing 25 ml of medium B. The fibroblasts continued dividing until confluency was reached, at which time the process was repeated until the desired passage level was reached. A passage level is defined as a division of the cell population into another culture vessel and does not denote the number of times a population doubles.

Fibroblast cultures were used experimentally between P-5 and

P-20 and cultured without antibiotics to ensure against unapparent (until taken off antibiotics) bacterial contamination and possible antibiotic interaction with experimental drug treatments. Fibroblasts were stored for

future use in liquid nitrogen between P-2 and P- 6 (as described below).

Method B

Method B was also used to establish primary fibroblast cultures. Dermal material was obtained during the establishment of epithelial cells (see below). The dermis was sliced into 1-3 mm^ segments in 0.5 ml 0.4% collagenase in PBS. Dermis from 4-6 foreskins was combined in 20 ml of 0.4% collagenase and dissociated by stirring for

2 hours at 37° C (51) . The resultant cell suspension was centrifuged for 5 minutes at 90 X g. The cellular pellet was added to 15 ml of medium B in a T-150 flask. After 24 hours the cells attached to the flask. The medium was removed and 25 ml of fresh medium B was added. Feeding and splitting of the cell cultures was done as described in method A. B. Establishment of epithelial cultures

Normal human neonatal foreskins were obtained under sterile conditions and stored in medium B for 2-3 days. The tissue was then washed twice with PBS and excessive dermal material was removed, which made subsequent processing easier. The tissue was stored for as long as 2 weeks in 5 ml of HBSS with antibiotics at 4° C. Epithelial cell cultures were established by a modification of the method of Yuspa et al.

(52). To obtain epithelial cells, 4-6 foreskins were removed from the storage containers and stretched dermal side down onto sterile Whatman #1 filter paper in a 100 mm petri dish. Ten mis of 0.25% trypsin was gently added to the bottom of the petri dish. The foreskins floated on the trypsin, exposing the dermal-epidermal junction to enzymatic digestion during an 18-24 hour incubation at 4° C. The

tissue was then removed from the trypsin and placed in a 1 0 0 mm petri dish containing 5 ml of PBS. The epidermis could then easily be stripped from the dermis and placed in the

PBS. The dermis was gently scraped to remove basal cells adherent to the basement membrane. The dermis was discarded or used for establishment of fibroblast cultures as described in method B (see above section). The PBS, which now contained the stratum corneum, stratum granulosum, basal cells and the basement membrane, was then sliced between two scapel blades to disrupt the epithelial tissue. This suspension was placed into a 15 ml centrifuge tube and a 5 ml PBS wash of the petri dish was also added. The suspension was pipetted up and down to further disrupt the tissue. After centrifugation at 90 X g for 5 minutes, the stratum corneum remained at the surface and was discarded.

The resulting precipitate, which contained the basal cells, was removed from the bottom of the centrifuge tube by a plastic pipet (Falcon, Cockeysville, MD) and placed in 15 ml of medium B in a collagen coated T-150 flask (the collagen coating technique is described below). Plastic pipets were used in all cell transfers, since cells are less adherent to plastic then glass pipets. A high density of epithelial cells (4-6 foreskins/flask) was necessary for good cell growth. The medium was replaced with 25 ml of fresh medium

B every 24 hours for one week, since the efficacy of the antibiotics was significantly decreased during the 24 hour incubation at 37° C. Epithelial cultures became confluent in 5-7 days and were split as described above for fibroblast cultures except that 0.1% trypsin with EDTA was used to remove the cells. Epithelial cultures were used experimentally at P-l since the culture conditions were not optimal for cell division and, therefore, with time the basal cells differentiated into non-dividing keratinocytes

(cells that produce keratin). Epithelial cultures at P-0 were also stored in liquid nitrogen as described below.

C. Collagen coating technique

Tissue culture plasticware was collagen coated since epithelial cells do not attach to untreated plastic effectively (51) . The 1% stock collagen, a gift from

Centerchem Co., NY, was diluted to 0.3% with PBS and stored at 4° C. Enough collagen was added to cover the bottom of the vessel and the excess was removed. The vessels were then placed in a humidified environment at 37° C for 1 hour, at which time the collagen had adsorbed to the plastic. The vessels were then washed gently with PBS and used immediately or an additional aliquot of PBS was added and the vessels were stored lightly capped for up to one week in the incubator.

D. Method for, counting cells.

Coulter Counter equipment and supplies

Coulter Counter, model #ZB1 Coulter Electronics, Inc. Hialeah, Florida

Isoton II coulter vials ( 2 0 mis) Curtin Matheson Scientific, Inc. (CMS)

Coulter Counter Procedure

Machine settings:

Amp. 2 Aperature current 1 Lower limit: 10 Upper limit: turned off

The Coulter Counter was turned on and allowed to warm up for

30 minutes prior to use. 9.5 mis Isoton II was gently placed in a coulter vial so that no air bubbles were introduced into the buffer. 0.5 ml of the trypsinized cell suspension was then carefully added into the Isoton solution. The vial was gently inverted three tiroes in order to mix the cells without introducing air bubbles, which can be inadvertently read as cells. Three counts per vial were performed and the average number was used to calculate the number of cells/ml by multiplying the obtained number by a predetermined factor. Since cell counts between

3,000-10,000 are the most accurate, any reading over 10,000 was corrected for cell coincidence in the orifice with a standard table.

Hemocytometer

An alternative method of counting trypsinized cells utilized the hemocytometer. After cells were dispersed into a single cell suspension, a 0.5 ml aliquot was added to an equal amount of trypan blue (1:2 dilution). Trypan blue was excluded from cells that were metabolically viable. The hemocytometer is divided into a grid in which the four outside and center areas were surveyed for cells. Both sides of the hemocytometer were counted, eliminating any cells that did not exclude the trypan blue dye, thereby giving a viable cell count. The number of cells/ml was determined by multiplying the number of cells counted times

the dilution factor (in this case 2 ) and multiplying by

10 . Cell counts of about 100 cells per chamber were the most accurate.

E. Cryostoraae of cells in culture Cryostorage of fibroblasts and epithelial cells was a convenient method for retaining viable stocks of cells for future experimentation. After either cell type was detached from a T-150 flask by trypsinization, the cells were pelleted by centrifugation at 90 X g for 5 minutes. The supernatant fraction was removed and the cells were resuspended in 1 ml of medium A with 10% dimethylsulfoxide

(DMSO), a cryoprotectant, and placed in a 2 ml sterile cryule vial (Wheaton, Millville, NJ). The vial was placed 2 inches above liquid nitrogen for 20 minutes. After this slow freezing process was completed, the vials were placed in liquid nitrogen storage. Upon removal of the vials from this storage, they were quickly placed in 37° C water or ethanol until the cells were thawed. The total thawing r=roceedure was completed in approximately 1 minute. The cells were placed i. - ot medium A and after 24 hours, viable cells were attached to the plastic or collagen coated flask. The medium was then removed and fresh medium A was added according to the proceedure described above. 2 0 II. PREPARATION AND TREATMENT OF CELLS__IN. CULTURE

FOR CYCLIC NUCLEOTIDE ASSAYS

A. Preparation of fibroblast cultures

Approximately one hundred 60 mm petri plates (Corning) were 5 inoculated with 4 X 10 fibroblasts/plate in 5 ml of

medium A and incubated at 37® C in a humidified 5% CO2 environment. After 24 hours, the medium was replaced with 3 ml of fresh medium A. Ninety-six hours after plating, the cultures were confluent and in plateau growth phase and the cyclic AMP to cyclic GMP ratio for these cells was at its highest level. Depending on the specific experiment, cultures were either monitored for cyclic nucleotide changes throughout this growth phase or were treated 96 hours after plating with various substances. The cyclic nucleotide content relative to protein was then determined as described below.

b . p.repa.r.a.tJi.Qn ..of epithelial-cuitures 5 Epithelial cells were plated at a density of 5 X 10 cells/collagen coated plate in 5 ml of medium B.

Twenty-four hours after seeding, the medium was removed and

3 ml of fresh medium B was added. Medium B was used in the experimental epithelial cultures, since cultures at P-l were not exposed to antibiotics for a sufficient duration to eliminate possible contaminating organisms. In order to parallel the fibroblast experiments, epithelial cultures 2 1 were monitored for their cyclic nucleotide concentrations throughout a 96 hour growth phase and were treated 96 hours after plating. All further protocols were identical for both fibroblast and epithelial cultures

C. UV-C Irradiation

The UV-C (150-290 nm) source was an 8 watt low pressure Hg vapor germicidal lamp (GE8T5) calibrated with a Latarjet meter (53) . Since the medium absorbs UV light it was removed prior to UV irradiation. The cells were exposed to

2 the UV source at a 2.5 J/m /sec fluence rate for 2-8 2 seconds (5-20 J/m ). The medium which was removed was then

replaced and the cultures were incubated for 0 - 6 hours.

Controls were treated identically except without the UV exposure.

D. Treatment of f ibr.obla5t_and_epithe.li.al. cultures

One hundred ul of the agent indicated in the figure legend for each experiment was added to five culture plates and incubated for the designated time interval. The medium was removed and was either discarded or frozen for later analysis of prostaglandins. The cells were washed twice with 0.15 M PBS, pH 7.2. Within 15 seconds after medium removal, 5% trichloroacetic acid (TCA) was added to the plates to halt cyclic nucleotide metabolism. The cells were scraped into a final volume of 2 ml of 5% TCA/plate. The precipitated protein was obtained by centrifugation at 1900

X g for 30 minutes. The protein fraction was made soluble 2 2 with 2 ml of 0.1 N sodium hydroxide and analyzed for protein content by the method of Lowry, et al. (54) as described below. The supernatant fraction was extracted three times with a 1:3 (v:v) ratio of sample to water-saturated ethyl ether to remove the TCA. Remaining ether was evaporated during a 10 minute incubation in a 57° C water bath. The pH of the samples was adjusted to 6.3 by the addition of 1M sodium acetate buffer pH 6.5 at a 1:10 (v:v) ratio of buffer to sample. The cyclic nucleotide content was determined by radioimmunoassay as described below.

e . P,,r..epflj^ jjp j_ a n d _ t r e,a tmffjQ.t o£ monocy-te g

Monocytes were obtained from the peripheral blood of normal donors by dextran sedimentation and ficcll-hypaque gradient centrifugation (55) . One million cells/150 ul/tube were incubated in medium A without FCS for one hour at 37® C in a

shaker bath prior to treatment. Ten ul of 250 uM 8 -MOP,

ETOH or water was added and after a zero to twenty minute incubation, 50 ul of 25% TCA was added to yield a final TCA

concentration of 6 %. The samples were further processed as described for the fibroblast and epithelial cultures.

f . Materials .

Reagents were obtained from the following: Haloperidol,

McNeil (Washington, PA.); dopamine, Regis (Morton Grove,

IL); 8 -MOP, TMP, IBX, isoproterenol, propranolol, histamine and tranylcypromine, 5 ’-guanylylimidodiphosphate, Sigma

Chemical Co., Inc. (St Louis, MO); forskolin, 23 Calbiochem-Behring Corp. (La Jolla, CA);

2',5'-dideoxyadenosine, P-L Biochemicals, Inc. (Milwaukee,

WI) .

FOR ENZYME ANALYSIS

A. Fibroblast Cultures for Enzyme. AnalysjLs.

Ten to fifteen sterile 150 mm glass petri dishes were seeded

with fibroblasts at a 1:2 split from T-150 flasks with 30 ml

of medium A. The medium was replenished every 2-3 days by

either adding an additional 15 ml or removing the old medium

and adding 30 ml of fresh medium A. The cells reached

confluency in approximately one week.

b. £jL0 P-^xatj.op_o£_.en?y?ngs

After the cells reached confluency, the medium was removed

from the plates and they were washed twice with 1 ml of 50 mM glycylglycine buffer (GCG), pH 7.6. The cells were

scraped with a rubber policeman into the residual GCG, an

additional 0.5 ml of GCG was used in a second scraping and

all cells were combined in a final volume of approximately 1 ml/plate and placed on ice. The suspension of cellular material was centrifuged in a Beckman L5-15B ultracentrafuge

for 1 hour at 100,000 X g at a temperature between 4 and

10" C. The resulting supernatant fraction contained the

soluble enzyme, phosphodiesterase, and was in the GCG buffer, which is compatible with the PDE assay system. The 24 resulting pellet contained the membrane bound enzyme, adenylyl cyclase. This pellet was resuspended in 25 mM Tris buffer and 5 mM M g C ^ then disrupted by careful homogenization with 7 strokes of a Dounce homogenizer. The

final volume was brought to 1 ml/original culture dish.

III. BIOCHEMICA L__ AN AL YTJ-CAL_TECHMXQnES

RADIOIMMUNOASSAY PROCEDURE

The cell culture samples were analyzed for cyclic AMP and cyclic GMP content by a modification of the radioimmunoassay

(RIA) procedure of Steiner et al. (56). To increase the sensitivity of the cyclic AMP and cyclic GMP RIA, 10 ul of a

2:5 mixture of acetic anhydride and triethylamine (v:v) was added to duplicates of the (100-300 ul) samples and standards (57). Cyclic AMP or cyclic GMP (Sigma) were 125 radiolabeled with I (New England Nuclear) using the procedures of Steiner et al., (56) or Miyachi et al. (58).

Labeled cyclic AMP or cyclic GMP were diluted in 0.25% bovine gamma-globulins (Sigma) to give 20,000-30,000 cpm per sample. The cyclic AMP and cyclic GMP antisera were developed in this laboratory and are highly specific and

sufficiently sensitive to measure 1 0 femtomoles of either nucleotide. The antiserum was diluted in 50 mM sodium acetate buffer, pH 6.3, with 0.2% bovine serum albumin

(Sigma) to provide a trace binding of 25-35%. After an 25

18-24 hour incubation at 4° C, the bound and free fractions

were separated by a 2 0 minute incubation with 60% ammonium

sulfate at room temperature. After a 30 minute centrifugation at 1900 x g, the supernatant fraction was

removed and the remaining precipitate was counted in a

Beckman 7000 gamma counter. The cyclic nucleotide concentrations were determined by a comparison with a 14 point standard curve.

PROTEIN ASSAY

A. Reagents for protein assay

Reagent A. 2% Na2 C03 in 0.10 N NaOH Reagent B. 0.5% CuSO^’Si^O in 1.0% Na/K tartrate Reagent C. 50 parts reagent A and 1 part reagent B Reagent D. Phenol reagent (Folin and Ciocalteau) was titrated against NaOH and was adjusted to 2N

Bovine serum albumin (BSA) (Sigma No. A-4503) was used for the preparation of the standard curve.

B. Protein Assay Method

Preparation of standards

Four hundred and eighty ul of 1 mg/ml stock was diluted with

720 ul 0.1 N NaOH to yield 80 g/200 ul. Using a serial 1:2

dilution with 0.1 N NaOH, a 6 point standard curve done in duplicate was constructed, including a NaOH blank which was used to zero the photometer. Absorbance was linearly proportional to the amount of protein from 5 to 80 ug/sample. Preparation of the samples the TCA precipitated cellular protein was solubilized in 0.1

N NaOH (2 ml for cells from a 60 mm petri plate). This volume of NaOH produced a protein concentration which was on the linear portion of the standard curve.

Assay proceedure

Either the standards or sample (200 ul) was aliquoted into

12 X 75 glass tubes. Two ml of reagent C was added and

mixed by vortexing, then incubated for 1 0 minutes at

25° C. One hundred ul of reagent D was added while simultaneously mixing, and the tubes were incubated for 30 minutes at 25° C. Absorbance at 580 nm was determined using a photometer.

ADENYLYL CYCLASE ASSAY

A. Principles of the adenylyl cyclase (AC) assay

In this assay system, adenylyl cyclase converts ATP to cyclic AMP which is quantitated by the cyclic AMP radioimmunoassay (described above). The enzyme was obtained from fibroblast cells in vitro as described above. The results of these assays were expressed as picomoles of cyclic AMP produced per mg protein per minute.

B. Reagents for the adenylyl cyclase assay

Buffer A

Buffer A contains 25 mM Tris, pH 7.8 and 5 mM MgClj and was stored at 4° C. This buffer was used for the 27

preparation of the adenylyl cyclase enzyme.

Buffer B

Buffer B contains the following compounds (Sigma):

50 mM Tris, pH 7.8 10 mM MgCl« 3 mM EDTA 9 mM Creatine phosphate 3 mM ATP 3 mM Dithiothreitol 0.6 mM Isobutylmethylxanthine 0.3 % BSA

Creatine kinase (Sigma) was added to yield a final

concentration of 3 units/tube. The first two components

listed in buffer B were twice the final concentration in the

reaction mixture while the remaining compounds were 3X the

final concentration. This buffer was made on the day of the

experiment from stock solutions. The ATP was shipped on dry

ice to eliminate possible degradation, and once diluted was

used immediately.

C. Assay Proceedure

The assay was run in a total volume of either 150 ul or 300 ul using either 50 ul or 100 ul of buffer B, respectively.

The various drugs tested were added in a volume which was one-third of the final incubation volume. The reaction was

started by the addition of the enzyme source (1 0 - 1 0 0 ug protein) in a volume which was one-third of the final reaction volume. Preliminary experiments indicated that the production of cyclic AMP was proportional to both the enzyme

concentration at an incubation time of 1 0 minutes and the reaction time between 0 and 20 minutes at 37° C. A heat

killed enzyme control tube was used as a blank. Its cyclic

AMP concentration was subtracted from that of the experimental tubes. The reaction was terminated by the addition of 0.2 M sodium acetate buffer at pH 4, followed by a 5 minute incubation at 90“ C in a water bath (59). The heat denatured protein was pelleted by a 30 minute centrifugation at 3,000 X g. The supernatant fraction was diluted 1:10 (v:v) with 50 mM sodium acetate buffer at pH

6.3, which optimized the pH for the subsequent cyclic AMP

RIA. For some drug treatments, additional 1:10 and 1:100

(v:v) dilutions were made to enable the cyclic AMP concentrations to be read from the cyclic AMP standard curve. The cyclic AMP RIA method and the protein assay method were as described above.

CYCLIC AMP PHOSPHODIESTERASE (PDE) ASSAY

A. Principles of the PDE assay

PDE converts cyclic AMP to 5 'AMP. Myokinase (EC 2.7.4.3) converts 5'AMP in the presence of very low concentrations of

ATP (which acts as a phosphate donor) to ADP. Pyruvate kinase (EC 2.7.1.40) converts ADP to ATP in the presence of the phosphate donor phosphoenol pyruvate (PEP). The resulting ATP concentration is directly proportional to the

5'AMP produced by PDE. ATP is then determined by the firefly luciferin-luciferase reaction by measurement of 29 phospholuminescence (60) . These reactions are summarized in

the equations below:

cyclic AMP — ^ — > 5'AMP (1) 5'AMP + ATP <— my-OM jIflSfi— > 2 ADp (2)

2 adp + 2 pep <-Pyyhvate-k.in.afi.e_ > 2 atp + 2 pyruvate (3)

ATP + luciferin + 02

AMP + PPi + oxyluciferin + CO2 + light (4)

B. Reagents for PDE assay

Reag£.nt_&.

150 mM glycylglycine buffer, pH 7.6 75 mM ammonium acetate 9 mM magnesium chloride 0.03 mM calcium chloride

0.7 8 mM phosphoenol pyruvate 15 mM dithiothreitol 3 nM ATP

This is 3 times the final concentration in the reaction

mixture. Reagent A is stable for several months at -4° C.

Reagent B

1 ml Reagent A 10 ul 3% BSA 10 Units myokinase (2.108 units/ul stock) 5 Units pyruvate kinase (1.596 units/ul stock) 3 ul of 10 mM calcium chloride

This is 3 times the final concentration in the reaction mixture. Reagent B is made on the day of each assay.

MOPS (morpholinopropane sulfonic acid) buffer

10 mM MOPS, pH 7.8 10 mM MgSO. 1 mM dithiothreitol 30

This reagent can be stored at 4° C for several months.

Luciferin-1uciferase reagent

1 ml MOPS buffer 15 mg luciferin-luciferase 0.5 % BSA

This reagent is made 1 hour prior to use, stored at 4° C, made in a plastic container and kept in the dark.

C. Source and Preparation of Reagents

M y .ofrinflS£

Myokinase from rabbit muscle (Sigma) was received as a

suspension in 3.2 M (NH4 )2 S0 4 solution, containing

0.001 M EDTA at pH 6.0 with an activity of 1085 units/mg protein. A unit is defined as follows: one unit will convert 2.0 umoles of ADP to ATP + AMP per minute at pH 7.6 and 37° C.

Eyr.u.y.flte-.kipase

Pyruvate kinase from rabbit muscle (Sigma) was received as a

suspension in 2.2 M (NH4 )2 S0 4 solution at pH 6.0 with an activity of 355 units/mg protein. One unit will convert

1.0 umoles phosphoenol pyruvate to pyruvate per minute at pH

7.6 at 37° C. Both myokinase and pyruvate kinase are centrifuged at 1000 X g for 10 minutes and the resulting precipitate, which contains the enzyme, was dissolved in reagent B.

Luciferin-luciferase

Luciferin-luciferase (Sigma, No. L-0633) contains luciferin, 31 luciferase, EDTA, glycine buffer salts and human albumin as a protein base.

D. Measurement of Light Generation

Light generation was detected by an integrating photometer

(SAI technology Co., model 3000). The instrument was modified to accept a repeating Hamilton syringe for microinjection. A plastic 1 ml syringe was substituted for the typical glass syringe, which eliminated the time dependent degradation of the light generating system.

E. Assay Proceedure

The assay was performed in 2 steps. In the first step, the

following components were added to a 6 X 50 mm glass tube:

Standard c.ur _v.e 50 ul h 2o 50 ul reagent B 50 ul various concentrations of 5'AMP (2.5-20,000 pmoles/50 ul)

SampLe-_t.uke.j5 50 ul enzyme sample 50 ul reagent B 50 ul cyclic AMP

The reaction was initiated with the addition of 5'AMP

(standard) or cyclic AMP (samples) after which the tubes were mixed. The samples were incubated for varying times at

37° C, placed in a boiling water bath for 10 minutes to stop the reaction, then cooled. In the final step, the ATP

concentration was determined by the addition of 2 0 ul of the firefly reagent and the subsequent light generated was detected by an integrating photometer. 32

PROSTAGLANDIN (PG) ASSAY

The concentration of PGE2 , PGF2ot, 6 -keto PGFlot (a stable metabolite of prostacyclin, PGI2) and thromboxane

(Tx) B2 (a stable metabolite of TxA2) in the cell culture medium was determined by radioimmunoassay. The PG

Tris buffer (50 mM Tris, 0.1% BSA, 0.05% sodium azide, pH

7.5) was used to dilute the media when concentrations exceeded those of the standard curve. Both standard and

labelled 6 -keto P G F ^ were stored in the PG Tris buffer,

while the standard and labelled PGE2 , PGF2oCand TxB2 were stored in 95% ethanol at -4° C. The PG standards (a gift from Upjohn) were made up at concentrations ranging from 0.1 to 10,000 picograms/0.1 ml aliquot. All of the tritium labelled (HTdR) PGs were obtained from New England

Nuclear. The specific activites for PGE2 , PGF2ot, 6 -keto

PGFlot and T x B 2 were 100-200, 150-180, 120-180 and

100-150 Curies/mmole, respectively. The labelled PGs were diluted in the appropriate solutions to give approximately

10,000 cpm/50 ul. The PG antibodies were developed in this laboratory (61) and were diluted in the PG Tris buffer to give a trace binding of 30-40% using 50 ul/sample. After an

18-24 hour incubation at 4° C, the bound and free fractions were separated by a 10 minute incubation with a 0.5 ml suspension of dextran-coated charcoal (250 mg Norit A, 25 mg

Dextran T-70, 100 ml PG Tris buffer) at 25° C. After a 20 minute centrifugation at 2500 X g (Beckman, J-6 B centrifuge)

the supernatant fraction containing the antibody bound PG

was decanted into a 3.5 ml scintillation vial. Scinti Verse

II (Fischer Scientific Co., Fairlawn, NJ) scintillation

cocktail (2.5 ml) in combination with the 0.7 ml of aqueous

sample produced a stable gel upon mixing which was counted

in a Beckman LS-7000. The results were expressed as the

percent of labelled PG bound versus the concentration of

unlabelled PG present. The sample PG concentrations were

determined by comparison with the standard curve and were

expressed as nanograms/ml of medium.

CYTOFLUOROMETRY OF HUMAN CELLS (62)

A. solutionsfor Acridine Orange Staining

Detergent solution

The detergent solution (0.08N HC1, 0.15 M NaCl and 0.1%

Triton x-100) was prepared by adding 0.88 g NaCl, 8 ml IN

HC1 and 0.1 ml Triton x-100 (100%) to a final volume of 100

ul with double-distilled H 2 O. This solution may be stored

at 4° C for at least 2 weeks.

Acridine_orange stock solution

The acridine orange stock solution (1 mg/ml) was made by

dissolving 50 mg of acridine orange in 50 ml of double

distilled water. This solution may be kept in the dark at

4° C for several months.

Citric acid buffer solution The citric acid buffer solution was prepared by mixing

184.25 ml 0.1 M citric acid with 315.75 ml 0.2 M

Na2 HPOij. This solution was stored at 4° C.

Acridine orange working solution

The acridine orange working solution was made by adding 1 ml

0.1 M EDTA and 0.6 ml acridine orange stock solution (1 mg/ml) to the citric acid buffer and bringing the final volume to 100 ml with the citric acid buffer. This solution may be stored for at least 2 weeks in the dark at 4° C.

B. A££i_djLne_Qrange. Staining for _Cytof luorometry

Three 60 mm petri plates containing treated and untreated

normal human fibroblasts were assayed every 1 2 hours throughout the 108 hour cell kinetic experiment. Removal of cells from the plates was done through trypsinization as previously described. For this proceedure 1-2 X 10® cells were needed. Therefore, the cells from three plates were combined in a 15 ml centrifuge tube and spun at 120 X g for

5 minutes. The supernatant fraction was removed and the cell pellet was fixed in 1 ml of 70% ethanol and stored at

4° C until the end of the 108 hour experiment. The fixed cells were centrifuged at 120 X g for 5 minutes and the ethanol was removed. The cells were washed once with distilled water and respun. The cell pellet was resuspended in 0.5 ml PBS and the cells were pipetted up and down approximately 15 times until a single cell suspension was obtained. A 0.2 ml aliquot of the cell suspension was 35 placed into a 15 ml round bottom plastic tube at 0° C. The cells were made permeable by the addition of 0.4 ml of the ice cold detergent solution. The cells were mixed well and incubated for at least 30 seconds at 0° C and 1.4 ml of ice cold acridine orange solution was added. The stained cells were kept on ice and the fluorescence was measured 15-30 minutes after the stain had been added. Cytofluorometry was performed by The Ohio State Comprehensive Cancer Center

Laboratory using a model 50 H cytofluorograph (Ortho,

Westwood, MA)

STATISTICAL ANALYSIS

Results were analyzed using Dunnett's t-test and Student's t-test and controlled for o< based on Bonfferoni inequality. RESULTS

THE EFFECT OF ULTRAVIOLET LIGHT ON CYCLIC AMP

The first observation that ultraviolet irradiation caused a change in cyclic AMP concentration in normal human fibroblasts in vitro was made in this laboratory (63).

These findings indicated that there was an increase in 2 cyclic AMP after 5-20 J/m of UV-C irradiation. Because of subsequent problems with reproducibility with various cell culture preparations, an examination of the cyclic nucleotide concentrations throughout a 108 hour growth phase was undertaken (64) . Figure 1 shows the cyclic AMP concentration profile throughout a 5 day period. In this experiment, 12 hours after plating, the cyclic AMP level was low. It increased slowly until a plateau was reached after

82 hours. The cyclic GMP profile is shown in figure 2. The cyclic GMP concentration, which was high initially, dropped to a constant low level by 72 hours. The ratio of cyclic

AMP to cyclic GMP was, therefore, low during the lag phase when cells were slowly dividing (12-48 hours), intermediate during the log phase when cells were rapidly dividing (48-82 hours) and high during the stationary phase when the cells reached confluency (82-108 hours). These results are in agreement with published data that demonstrate the

36 FIGURE 1. THE EFFECT OF TIME IN CULTURE ON THE CYCLIC AMP CYCLIC THE ON CULTURE IN TIME OF EFFECT THE 1. FIGURE CYCLIC RMP (PICOMOLES/MG PROTEIN) 10 12 2 8 6 4 ± the standard deviation for deviation standard the ± nucleotide metabolism. The concentration of concentration The metabolism. nucleotide the mean concentration of cyclic AMP/mg protein AMP/mg cyclic of concentration mean the described as made were measurements protein and and PBS with twice washed quickly were plates CO 5%a FIBROBLASTS HUMAN NORMAL OF CONCENTRATION ntemtos eto. ahpit represents point Each section. methods the in radioimmunoassay by determined was AMP cyclic 4X at seeded were fibroblasts human Normal rae ih5 TAt ihbtfrhr cyclic further inhibit to TCA 5% with treated in C 37° at incubated and plate cells/60mm 10 niae ie, h eimws eoe, the removed, was medium the times, indicated 1 ______j _ 2 4 6 8 0 2 4 6 108 96 84 72 60 48 36 24 12 t 1 HOURS AFTER PLATING AFTER HOURS 5 ------uiiidevrnet A the At environment. humidified ______1 ------1 ______1 ------1 ______1 ------1 ______1 ------1 ______0 1 plates. 1 ------1 ______1 ------1 ______

r L_

FIGURE 2. THE EFFECT OF TIME IN CULTURE ON THE CYCLIC GMP CYCLIC THE ON INCULTURE TIME OF EFFECT THE 2. FIGURE CYCLIC GMP (PICOMOLES/MG PROTEIN) .2 .4 .1 .3 .5 .6 .7 .8 .9 12 h cls ee rae s ecie i the in described as treated were cells The ocnrto f ylcGPb radioimmunoassay. by GMP cyclic of concentration CONCENTRATION OF NORMAL HUMAN FIBROBLASTS HUMAN NORMAL OF CONCENTRATION eedo figure of legend 24 HOURS AFTER PLATING AFTER HOURS 66 84 60 36 48 1 n nlzdfr their for analyzed and 72 6108 96

39 relationship between cyclic AMP and cyclic GMP concentration and cell density (65,66) . In view of these results, the

2 cells were irradiated with a fluence of 10 J/m UVC light either in the log phase of cell growth (about 44 hours after plating) or in the plateau phase (96 hours after plating).

The cyclic AMP concentration in irradiated cells was unaltered in log phase cultures (figure 3), but in confluent cultures 2 and 4 hours after irradiation the cyclic AMP concentration was increased over controls by 43% and 90%, respectively (figure 4) . Although cyclic GMP measurements were made on this and all subsequent experiments, no effect of UV light on the concentration of this compound was detected. In order to verify that this difference in response was due to the confluency of the cells and not to nutritional factors, cells were seeded either at the normal 5 density (4 X 10 cells/plate) or at a lower density (4 X 3 10 cells/plate). Half of the cells in each group were then given fresh medium 24 hours prior to UV treatment.

Whether or not they had recieved fresh medium, cultures seeded at a low density (which had not reached confluency) were unaffected by UV light, while cultures seeded at a higher density (and which were confluent) showed a rise in cyclic AMP after irradiation (data not shown). On the basis of this experiment we concluded that the response of the cells to UV irradiation depends either on the level of cyclic nucleotide concentration or the degree of confluency FIGURE 3. THE EFFECT OF ULTRAVIOLET IRRADIATION ON THE ON IRRADIATION ULTRAVIOLET OF EFFECT THE 3. FIGURE CYCLIC AMP (PICOMOLES/MG PROTEIN) 10 12 14 2 8 4 6 0 ewe ons ihr ihn r between or within either difference points statistical nobetween was There plates. -et r h tdn' t-test. Student's the or t-test Dunnett's the by determined as groups treatment 3-5 of deviation standard the ± concentration experiments, each point represents the mean the represents point each experiments, («) not or /second J/m 2.5 of rate a at nm) either irradiated with with irradiated either grown and zero time at plated were Fibroblasts PLATING AFTER 44 HOURS FIBROBLASTS HUMAN OF CONCENTRATION AMP CYCLIC the times indicated. In this and subsequent and this In indicated. times the irradiated (O) • The concentration of cyclic AMP cyclic of concentration (O) The irradiated • in the cells was determined after irradiation at irradiation after determined was cells thein n utr fr 4 or. h^el ee then were The^cells hours. 44 for culture in 1 2 HOURS IQ IQ / U ih (254 light UV J/m 3 4

FIGURE 4. THE EFFECT OF ULTRAVIOLET IRRADIATION ON THE ON IRRADIATION ULTRAVIOLET OF EFFECT THE 4. FIGURE CYCLIC RMP CPICOMOLES/MG PROTEIN) 4 - 0 were significantly different (p C.005) from the from (p C.005) different significantly were were irradiated irradiated were orsodn o-raitdcnrl (Student's controls non-irradiated corresponding UV after points time All plating. after hours t-test). the in described as treated were cells The PLATING AFTER HOURS 96 FIBROBLASTS HUMAN OF CONCENTRATION AMP CYCLIC eedo fgr 3 ecp ta tee cultures these that except 3, figure of legend irradiation were significantly different significantly were irradiation (Dunnett's t-test). The 2 and 4 hour time points time 4 hour and 2 The t-test). (Dunnett's control time zero irradiated the from (p<.005) 1 ( k 2 ) r o irdae (O) 96 irradiated not or HOURS 4 3

42 of the cells prior to treatment.

The rise in cyclic AMP was proportional to the dose of UV irradiation as measured six hours after UV treatment (figure

5). The concentration of cyclic AMP was increased over basal level by 5%, 52% and 102% with a UV dose of 5, 10 and 2 20 J/m , respectively. To explore the effects of UV exposure beyond the cyclic AMP dose and time dependent responses, agents that were known to interact with UV light were examined.

THE EFFECT OF UV LIGHT AND PABA ON CYCLIC AMP

Para-aminobenzoic acid (PABA) absorbs light below about

330 nm (67) and thus effectively blocks UVB and UVC transmission. PABA was tested as a blocker of the in vitro

UV-mediated increase in cyclic AMP concentration (figure 6 ).

In the absence of PABA, the cyclic AMP concentration was 76% 2 greater than control six hours after a 10 J/m UV exposure. Pretreatment of the cells with increasing concentrations of PABA progressively decreased the effect of irradiation on cyclic AMP concentration, until 0.5 mM where the UV effect was totally blocked. Under identical circumstances, the lower concentrations of PABA alone did not alter the cyclic AMP concentration, but at concentrations of 0.5 and 1 mM PABA caused a slight increase over control levels. These results indicate that the change in cyclic AMP was a direct consequence of the interaction of FIGURE 5. THE DOSE DEPENDENT EFFECT OF UV IRRADIATION ON IRRADIATION UV OF EFFECT DEPENDENT DOSE THE 5. FIGURE CYCLIC AMP (PICOMOLES/MG PROTEIN) 20 2 2 10 2 1 16 IB 8 Analysis by Dunnett's t-test indicated that UV that indicated t-test Dunnett's by Analysis controls. THE CYCLIC AMP CONCENTRATION OF FIBROBLASTS OF CONCENTRATION AMP CYCLIC THE oe o 0ad2 / wr significantly were 20J/m and 10 of doses Fibroblasts were grown to confluency and confluency to grown were Fibroblasts cyclic AMP was determined by radioimmunoassay. by determined was AMP cyclic ultraviolet light. The cells were then were cells The light. ultraviolet incubated for incubated of dose indicated the with irradiated pC05 dfeet rmte unirradiated the from different (pC.005) 1 I 20 IS 10 S 6 VDO (J/M2) E OS D UV hours and their concentration of concentration their and hours ••

FIGURE FIGURE CYCLIC AMP (PICOMOLES/MG PROTEIN) 0 1 2 1 14 16 2 4 6 8 6 TE FETO AAO H VIDCDRS IN RISE UV-INDUCED THE ON PABA OF EFFECT THE . minutes and were either irradiated with with irradiated either were and minutes were incubated for an additional additional an for incubated were irbat ee rae ih h indicated the with treated were Fibroblasts J/m UV (») or not irradiated (O). The plates The (O). irradiated (»)not or UV J/m PABA pretreatment (Dunnett's t-test). (Dunnett's pretreatment without PABA control irradiated the from (p<„005)different significantly were concentrations ocnrtoso aaaioezi cdfr 30 for acid para-aminobenzoic of concentrations AMP CYCLIC rtetetwt 5 u AAo greater or PABA uM 250 with Pretreatment content* AMP cyclic their for analyzed then j 1 2 4 10 0 40 1000 400 200 100 40 20 10 0 — / / — t t i fRlfMNBN0C CD CUM) ACID PflRfl-flMIN0BENZ0IC _ i . j i "" i 1 i '"i"i 'i - i . j j ______i i i » ------—i i i 1 i n i i i i 1— 1 1 1 »i» 111111 » 6 hours and hours ------—s r s— 1—

0 1

•* _

UV light with the cells. In view of the fact that a

UV-absorbing compound could block the UV-induced effect, we

next determined whether the UV effect was enhanced by an agent which is known to interact with the effects of UV

light.

THE EFFECT OF UV LIGHT AND 8 -MOP ON CYCLIC AMP

A number of UV effects in vitro are augmented by

8 -methoxypsoralen (8 -MOP). Its effect on human cells in vitro was tested both with and without UV irradiation -

(figure 7). Following a 30 minute incubation with ethanol

(the vehicle control) or increasing concentrations of 8 -MOP, o cultures were either irradiated with 10 J/m of UVC light or were used as unirradiated controls. The cyclic AMP and

cyclic GMP concentrations were determined 6 hours after

irradiation. The cyclic GMP concentration of the cells after treatment did not change significantly from control

(data not shown). As shown previously, UV irradiation caused an increase in cyclic AMP concentration over control

levels. Unexpectedly, increasing concentrations of 8 -MOP in the absence of UV light caused a dose dependent increase in cyclic AMP concentration. The combined treatment of 500 uM

8 -MOP and UV irradiation produced an increase in cyclic AMP which was 357% over ethanol controls, and was the

approximate sum of each treatment effect (1 0 2 % increase for

UV irradiation and 195% increase for 500 uM 8 -MOP FIGURE 7. THE EFFECT OF OF EFFECT THE 7. FIGURE CYCLIC AMP (PICOMOLES/MG PROTEIN) 20 25 10 15 Analysis by Dunnett's t-test indicated that indicated t-test Dunnett's by Analysis by Analysis control. a as ethanol with The cells not treated with with treated not cells The concentration. AMP cyclic their for analyzed (^) not or light UV J/m 10 to exposed were cells treated with with treated cells the that indicated t-test Student's cells pretreated with with pretreated cells treated cells were different (p <.005) from the from (p<.005) different were cells treated to prior hours 96 £or incubated were Fibroblasts raito r dfeetfo oto level control from different are irradiation irradiated (X). Six hours later the cells were cells the later hours Six (X). irradiated ramn. fe a3 iue ramn ih the with treatment minute 30 a After treatment. FIBROBLASTS IN CONCENTRATION AMP CYCLIC indicated concentrations of concentrations indicated (* p <.01 and ** p <.005). p ** and <.01(* p j 0 // t i i — i— r i i r i— -EHXP0RE (UM) 8-METH0XYPS0RRLEN 0 0 0 0 200 100 40 20 10 * i i i i i i m i i 8 11 -MOP AND UV IRRADIATION ON THE ON IRRADIATION UV AND -MOP 8 -MOP with and without UV without and with -MOP 8 MPadte irradiated. then and -MOP i i i j i i —i i i— n t— 8 -MOP were treated were -MOP 8 -MOP, the plates the -MOP, 1 1 1 11 8 «« -MOP 400

L J t I

—

r 46

47 treatment).

Theoretically, the wavelength of UV light used in this experiment does not photoactivate the psoralen molecule.

Therefore, any effects due to their combined presence should be independent. To test this, the order of UV irradiation

and 8 -MOP exposure was reversed. When cells were treated

with increasing doses of 8 -MOP, either before or after UV

irradiation, there was an identical 8 -MOP dose dependent

increase in cyclic AMP concentration after 6 hours (data not shown). The increase seen was the sum of the independent UV

and 8 -MOP treatment effects. The effect of these treatments at shorter incubation times also supported independent effects. At 1, 2 and 4 hours after treatment with 500 uM

8 -MOP or 8 -MOP in combination UV the effects on cyclic AMP concentrations were indistinguishable. Both treatment regimes increased cyclic AMP by 170% over control after 1 hour which decreased to 108% and 56% by 2 and 4 hours,

respectively. It is, therefore, only after the 6 hour treatment that the increasing UV time dependent effect and

the decreasing 8 -MOP time dependent effect become additive when the treatments are combined. During these experiments,

it became apparent that the 8 -MOP effect on cyclic AMP occurred rapidly (within one hour). Subsequently, shorter incubation times were examined. 48

THE EFFECT OF 8 -MOP ON CYCLIC AMP

8 -MOP alone (500 uM) caused a rise in cyclic AMP within

one minute and reached a maximum between 5 and 10 minutes

(figure 8 ). For technical reasons, the 8 -MOP dose response

(figure 9) was performed at 15 minutes. In this experiment,

concentrations of 8 -MOP up to a maximum of 250 uM caused a

dose dependent rise in cyclic AMP concentration. This

demonstrated that 8 -MOP led to a rise in cyclic AMP after

short incubation times as well as the longer incubation

times.

Although there is no measureable UVA light (364 nm)

emitted from yellow-fluorescent room lights, and the

previous results indicated that the effect of 8 -MOP on

cyclic AMP was independent of its interaction with UV light,

the possibility that some interaction was responsible for

the cyclic AMP effect had not been entirely eliminated.

Therefore, time and dose response experiments were carried

out in the absence of normal room light (figures 1 0 and 1 1 ).

A Kodak red safety light allowed manipulation of the culture plates while ensuring that extraneous UVA, B and C would not photoactivate the psoralen molecule. The resulting increase in cyclic AMP was significant within one minute of addition

of 250 uM 8 -MOP and remained elevated for at least one hour.

The dose dependent increase in cyclic AMP was similar to the previous dose response done in room light (figure 9). As in FIGURE FIGURE CYCLIC BMP CPICOMOLES/MG PROTEIN) 12 14 0 1 16 0 2 6 8 4 8 . THE TIME DEPENDENT EFFECT OF OF EFFECT DEPENDENT TIME THE . Analysis by Student's t-test indicated that indicated t-test Student's by Analysis AMP CONCENTRATION OF FIBROBLASTS OF CONCENTRATION AMP Dunnett's t-test indicated that the treated the that indicated t-test Dunnett's Ninety-six hours after plating, fibroblasts were fibroblasts plating, after hours Ninety-six times after one minute. one after times points. all at treatment second 15 the from .005) different (p< significantly were cells rae ihrwt 20 uM 250 with either treated determined by radioimmunoassay. Analysis by Analysis radioimmunoassay. by determined was cells the in AMP cyclic times, of indicated the concentration At the (A). alone ethanol or 8 .005) different from ethanol control at all at control ethanol from different .005) MPtetdclswr infcnl (p< significantly were cells treated -MOP 10 525 15 MINUTES 20 8 MP nehnl (x) ethanol in -MOP 8 -MOP ON THE CYCLIC THE ON -MOP 30S

FIGURE 9. THE DOSE DEPENDENT EFFECT OF OF EFFECT DEPENDENT DOSE THE 9. FIGURE CYCLIC RMP (PICOMOLES/MG PROTEIN) 10 12 2 4- 6 8 - - - - Analysis by Dunnett's t-test indicated that indicated t-test Dunnett's by Analysis M CNETAINO FIBROBLASTS OF CONCENTRATION AMP tao control. ethanol respectively. protein, AMP/mg cyclic picomoles significantly (p < .005) different from the from different .005) (p < significantly were levels ethanol and the with minutes 15 for treated were Fibroblasts 8 niae ocnrtos of concentrations indicated -MOP concentrations at and above 31.3pM were 31.3pM above and at concentrations -MOP 5 0 0 0 0 200 100 50 20 10 5 0 1 * ------1 ----- 1 ---- METH S0RR (UM) N E RL R 0 PS Y X H0 T E -M 8 T' I I I I ~ 'l T I j 1 T i i i i i i i i i i i 8 . 6 ---- «« —f~l I I I I I l ~ f 1— ± 0.3 and 7.7 ± 0.4 ± 7.7 and 0.3 ± 8 -MOP. The basal The -MOP. 8 -MOP ON THE CYCLIC THE ON -MOP «« L I J t --- «*

1 r —

FIGURE 10. THE TIME DEPENDENT EFFECT OF EFFECT DEPENDENT TIME THE 10. FIGURE CYCLIC AMP (PICOMOLES/MG PROTEIN) 0 1 2 1 14 16 IB 0 2 6 4 8 1 1 2 2 3 3 4 4 5 5 60 55 50 45 40 35 30 25 20 15 10 5 UNDER RED LIGHT RED UNDER TREATED WHEN FIBROBLASTS OF CONCENTRATION AMP tdn' tts idctdta te nrae in increase the that indicated t-test Student's one minute. one 4ffer points time all at control ethanol from different (p<.005) significantly was AMP cyclic Kodak red safety bulb for light. Analysis by Analysis light. for bulb safety red a Kodak only using times indicated the (A) for ethanol uM 250 with trated were Cells MINUTES 8 -MOP ON THE CYCLIC THE ON -MOP 8 MP (*) or -MOP

FIGURE II. THE DOSE DEPENDENT EFFECT OF OF EFFECT DEPENDENT DOSE THE II. FIGURE CYCLIC RMP CPICOMOLES/MG PROTEIN) 10 12 14 2 4 6 6 - 0 ehd. nlssb unt' tts indicated t-test Dunnett's by Analysis methods. UNDER RED LIGHT RED UNDER AMP CONCENTRATION OF FIBROBLASTS WHEN TREATED WHEN FIBROBLASTS OF CONCENTRATION AMP Kodak red safety bulb for light. Analysis for Analysis light. for bulb safety red Kodak nrae oto s niae (**<.005). p indicated as control untreated concentrations of concentrations increasing with treated cells that the with 15minutes for treated were Cells cyclic AMP was carried out as described in the in described as out carried was AMP cyclic indicated concentrations of concentrations indicated A V T ---- i ij i -EHXPOfLN (UM) 8-METHOXYPSORflLEN 1 --- 1 -- 5 0 100 50 25 —I1— 1111 j i I I 8 -MOP were different from the from different were -MOP T 8 -MOP using only a only using -MOP 8 -MOP ON THE CYCLIC THE ON -MOP J iiJ i i l l 1 ---

1 -- 250 1 — 1 I I I

all previous 8 -MOP experiments, cyclic GMP concentrations

were unaltered compared to control in the 8 -MOP time and

dose experiments (figures 12 and 13).

To this point, all 8 -MOP experiments utilized confluent

cultures. Since the UV-mediated effect on cyclic AMP

concentration was dependent on the degree of confluency of

the culture, the effect of 8 -MOP on cells at various times

after plating was tested. Cells which had been plated for

48, 72 and 96 hours were treated with increasing doses of

8 -MOP for 15 minutes (figure 14). Although the control

concentration of cyclic AMP varied with time in culture (3.6

pmoles/mg protein at 48 hours to 6.5 at 96 hours), the 8 -MOP

dose dependent increases in cyclic AMP at different stages

of confluency were essentially parallel. Therefore, the

8 -MOP mediated effect was independent of state of confluency

of the culture.

THE LONG TERM EFFECT OF 8 -MOP TREATMENT ON FIBROBLASTS

The effect of long term treatment with 8 -MOP on cyclic

nucleotide concentrations, protein synthesis, cell toxicity

and DNA profiles were examined. Fibroblasts were plated in

the usual manner and thereafter, 5 plates per treatment

group were removed every 1 2 hours for 1 2 0 hours for cyclic nucleotide and protein determinations and 3 plates per group were used for determination of cell number and DNA profile analysis. Treatment of cultures was initiated 48 hours FIGURE 12. THE TIME DEPENDENT EFFECT OF EFFECT DEPENDENT TIME THE 12. FIGURE CYCLIC GMP (PICOMOLES/MG PROTEIN) .1 2 . .3 .4 .5 0 1 1 2 2 3 3 4 4 5 5 60 55 50 45 40 35 30 25 20 15 10 S no difference in cyclic GMP concentrations after concentrations GMP cyclic in difference no by Analysis light. for bulb safety red Kodak UNDER RED LIGHT RED UNDER TREATED WHEN FIBROBLASTS OF CONCENTRATION GMP controls and the zero time control. time zero the and controls tdn' -etadDnetstts indicated t-test Dunnett's and t-test Student's Cells were treated with 250 uM uM 250 with treated were Cells ethanol (O) for the indicated times using only a only using times indicated the (O) for ethanol 8 -MOP treatment when compared to ethanol to compared when treatment -MOP MINUTES 8 -MOP ON THE CYCLIC THE ON -MOP 8 MP (x) or -MOP

FIGURE 13. THE DOSE DEPENDENT EFFECT OF OF EFFECT DEPENDENT DOSE THE 13. FIGURE

CYCLIC GMP (PICOMOLES/MG PROTEIN) .2 .3 - .4- .5 - 1 - - UNDER RED LIGHT RED UNDER TREATED WHEN FIBROBLASTS OF CONCENTRATION GMP t-test. Dunnett's the using detected were significant No differences previously. described cyclic GMP concentrations were carried out as out carried of were Analysis concentrations GMP cyclic light. for bulb safety red Kodak the with minutes 15 for treated were Cells indicated concentrations of concentrations indicated I I I I J -EHXP0RE (UM) 8-METH0XYPS0RRLEN 5 0 100 50 25 1 1 1 ! 8 -MOP using only a only using -MOP 8 i T -MOP ON THE CYCLIC THE ON -MOP * » 1 ___ ---

I « I I » « I 1 —l l— l I — I' 250

FIGURE 14. THE EFFECT OF OF EFFECT THE 14. FIGURE CYCLIC RMP (PICOMOLES/MG PROTEIN) 0 1 12 14 2 4 6 8 ocnrto (unt' t-test). (Dunnett's concentration culture, in time the of Independent previously. treatment with 62.5 u or more more or u 62.5 with treatment oe of doses significant (p C.005) increase in cyclic AMP cyclic in increase (p C.005) significant described as AMP cyclic of concentration the ru o clsws nuae ih increasing was* with cells of incubated group X 4 of a.concentration at plated were Cells PLATING AFTER TIMES DIFFERENT AT CULTURES 0 1 6 or. t 8 □, 2 O ad 6 x hus a (x) hours, 96 (O) and (□), 72 48 At hours. 96 0 cells/plate and grown in culture for up to up for culture in grown and cells/plate 8-METHOXYPSORflLEN 8-METHOXYPSORflLEN < ►— 8 -MOP for 15 minutes and analyzed for analyzed and minutes 15 for -MOP 550 25 8 -MOP TREATMENT ON FIBROBLAST ON TREATMENT -MOP CUM) 100 8 -MOP caused a caused -MOP 250

after plating. Figure 15 illustrates the cyclic AMP profile of untreated cells throughout the entire growth period along

with the ethanol control and 250 uM 8 -MOP treated cultures starting 48 hours after plating. The cyclic AMP

concentrations were also measured at 0.5, 1, and 6 hours after treatment to reconfirm the rapid increase mediated by

8 -MOP treatment. The cyclic AMP concentration in the untreated cells increased with time in culture. After

treatment with 8 -MOP, the cyclic AMP concentration remained high for one hour and decreased progressively during the next 24 hours, at which time it became equal to the concentration in untreated cultures. Thereafter, the cyclic

AMP concentration remained the same between the various treatment groups. Figure 16 illustrates the cyclic GMP profile of the cultures described for figure 15. The cyclic

GMP concentration in the untreated cells decreased with time

in culture. Conversely, after treatment with 8 -MOP, the cyclic GMP concentration remained constant throughout the 72 hour incubation. When the cyclic AMP to cyclic GMP ratio was plotted, a slightly different pattern emerged (figure

17). The ratio pattern for untreated cultures was typical for growing fibroblasts in vitro. Ethanol treatment inhibited the increase seen in untreated cultures for the first 24 hours and thereafter the ratio paralleled, but was lower then that of the untreated cultures. Following

treatment with 8 -MOP there was a spike in the cyclic AMP/GMP FIGURE 15. THE EFFECT OF EXTENDED INCUBATION WITH WITH INCUBATION EXTENDED OF EFFECT THE 15. FIGURE CYCLIC AMP (PICOMOLES/MG PROTEIN) 10 2 4 6 8 lts nec ru eeaaye o their for analyzed were group each in plates a significant (p <.005) difference in cyclic AMP cyclic in difference indicated (p<.005) t-test Student's significant by a Analysis control. FIBROBLASTS OF CONCENTRATION AMP CYCLIC THE cyclic AMP concentration. Analysis by Dunnett's by Analysis concentration. AMP cyclic controls. after hours 24 first the for concentrations and 1 0.5, at difference culture in hours 48 After cells/plate. 10 4 X of concentration a at plated were Fibroblasts tao aoe (O) uM 250 alone with ethanol , treated or (□),with treated untreated were cells the -et niae sgiiat (p<•005) significant a indicated t-test ramn cmae o h zr ie treatment time zero the to compared treatment 6 8 1, 0.5, 0, for treatment (x) After ethanol in . 12 n 1 or n vr 2husteefe, 5 thereafter, hours 12 every and hours 12 and -MOP treatment when compared to the ethanol the to compared when treatment -MOP 436 24 OR ATR PLATING AFTER HOURS 48 60 72 6 hus after hours 49 108 96 84 8 8

-MOP ON -MOP -MOP 0 2 1

-MOP

FIGURE 16. THE EFFECT OF EXTENDED INCUATION WITH WITH INCUATION EXTENDED OF EFFECT THE 16. FIGURE CYCLIC GMP (PICOMOLES/MG PROTEIN) 1.2 1.4 1.6 1.8 .2 .4 .6 .8 1 2 3 4 6 7 8 9 18 120 108 96 84 72 60 48 36 24 12 0 1

ih tao (O) ethanol with , uM 250 FIBROBLASTS OF CYCLIC CONCENTRATION GMP THE of cyclic GMP. cyclic of figure in 15 described as incubated were Cells nrae () n nlzdfr h concentration the for analyzed (□) and untreated J t ______I 1 ______---- I 1 ______---- OR ATR PLATING AFTER HOURS I 1 ______---- I 1 ______---- I 1 ______---- 8 MP x o left (x) or -MOP I 1 ______---- I 1 ______---- I 1 ______8 ---- -MOP -MOP ON

L r 59

FIGURE 17. THE EFFECT OF EXTENDED INCUBATION WITH WITH INCUBATION EXTENDED OF EFFECT THE 17. FIGURE CYCLIC RMP/CYCLIC GMP 0 6 - ih tao (O) ethanol with THE RATIO OF CYCLIC AMP TO CYCLIC GMP CYCLIC TO AMP CYCLIC OF RATIO THE ylcGPws acltdfo h rsls shown results the from to calculated AMP cyclic was of GMP ratio cyclic The (o). untreated Cells were incubated as described in figure 15 figure in described as incubated were Cells n iue 1 ad 16. and 15 figures in 12 24 36 OR ATR PLATING AFTER HOURS 46 , 5 uM 250 072 60 8 MP (») left or -MOP 84 6 0 120 108 96 8 -MOP ON -MOP —i:

0 6

ratio which returned to control levels within 1 2 hours and then remained constant for an additional 60 hours. Figure

18 shows the increase in cell density throughout the culture 5 period. Cells were seeded at 4 X 10 cell/plate and 12 5 hours later 2.25 X 10 cells/plate were attached to the culture dish (56% plating efficiency). After 120 hours in g culture there was an average of 2.1 X 10 cells/plate.

Treatment with either ethanol or 8 -MOP inhibited the increase in the cell number seen in the untreated cultures.

Seventy-two hours after ethanol and 8 -MOP treatment, the cultures had 1.2 X 10^ cells/plate (one half that of the untreated cultures). This was not due to cell death, since

99% of the cells excluded trypan blue. No morphologic differences between untreated and treated cultures could be detected by microscopic visual examination of the cultures.

When cells die and detach from the culture vessel, some cellular material remains attached to the plastic. These

"footprints" could not be detected in any of the cultures.

Figure 19 shows the ethanol and 8 -MOP effect on accumulation of protein in cells in culture. The total protein in untreated cells increased by 107% between 48 and 120 hours.

The pattern of protein accumulation in ethanol and 8 -MOP treated cultures was not identical. Total protein increased

by 79% in ethanol treatd cells and by 45% in 8 -MOP treated cells, even though these two treatment groups had the same number of cells. Therefore, the ethanol treated cells may FIGURE 18. THE EFFECT OF EXTENDED EXPOSURE TO EXPOSURE EXTENDED OF EFFECT THE 18. FIGURE CELL NUMBER PER PLRTE (X 1 0 s ) 1 2 3 4 6 7 8 9 18 120 108 96 84 72 60 48 36 24 12 0 ee eemnduigacutr one as counter coulter a using determined were numbers cell and group each from removed were the untreated controls. untreated and the cultures treated the between treatment after significant a was there However, cultures. elnme bten tao and ethanol between number in cell difference significant no indicated t-test NUMBER CELL FIBROBLAST described in methods. Analysis by Student's by Analysis methods. in described iue1. t h idctdtms 3plates times, indicated the At 15. figure (O) ethanol with , uM 250 treated were Cells 8 p <05 dfeec i el ubr 6 hours 36 number cell in difference (p<.005) MP k o lf utetd □ a dsrbd in (□) described as untreated left (k) or -MOP OR ATR PLATING AFTER HOURS 1 i 8 -MOP treated -MOP 8 -MOP ON -MOP

2 6

FIGURE 19. THE EFFECT OF EXTENDED EXPOSURE TO EXPOSURE EXTENDED OF EFFECT THE 19. FIGURE PROTEIN (UG/PLATE) 200 250 100 300 350 150 1 2 3 4 6 7 8 3 18 120 108 36 84 72 60 48 36 24 12 0 tdn' tts idctda significant a indicated t-test Student's content as described in methods. Analysis by Analysis inmethods. described as content INCULTURE FIBROBLASTS OF CONTENT PROTEIN different from untreated cultures. untreated from different el ee rae ih tao (O)r uM 250 ethanol with treated were Cells utrs 6 or fe tetetwt either with treatment after hours 36 cultures after hours 72 and iue1. t h idctdtms 5plates times, indicated the At 15. figure to the ethanol controls. The protein content of content protein The controls. ethanol the to from each group were analyzed for their protein their for analyzed were group each from 8 8 p .0) ifrne npoenpr lt t 60 at plate per inprotein difference (p <.005) MPo ehnlwr sgiiaty (p<.005) significantly were ethanol or -MOP MP * o lf utetd H a dsrbd in (H) described as untreated (*) left or -MOP OR ATR PLATING AFTER HOURS 8 -MOP treatment when compared when treatment -MOP 8 -MOP ON THE ON -MOP

be more metabolically active then those treated with 8 -MOP.

DNA profiles were also examined using acridine orange DNA

staining and monitoring green fluorescence with a

cytofluorograph. The pattern of fluorescence is

representative of the number of cells with 2N DNA (cells in

G^) and 4N DNA (cells in G 2 ). The DNA fluorescence

patterns for untreated, ethanol and 8 -MOP treated cells

after 12 hours are similar (figure 20). The only difference

in pattern occured 36 hours after treatment (figure 21). In

the 8 -MOP treated cells, the lack of the characteristic

large, well defined peak suggests that the cells had been

blocked and are progressing from G^ through S phase and

THE EFFECT OF 8 -MOP ON CYCLIC AMP IN EPITHELIAL CELLS AND

MONOCYTES

In order to determine if the effect of 8 -MOP on cyclic

AMP was specific for fibroblasts, epithelial cells and monocytes were tested in vitro for their responsiveness to

8 -MOP. As detailed in the methods, epithelial cells were obtained from normal human foreskins and established in culture. Using a protocol similar to the one used for the fibroblast cultures, the time and dose dependent effect of

8 -MOP on cyclic AMP was determined. Within one minute after

addition of 8 -MOP the cyclic AMP concentration was increased slightly and by 15 minutes it was 182% over control (figure FIGURE 20. THE DNA FLUORESCENCE PATTERN 12 HOURS AFTER 8-MOP TREATMENT COMPARED TO CONTROLS

Cells were obtained from the plates used to determine cell numbers (figure 18), then fixed and stained as described in the cytofluorometry methods. Profile A represents untreated cultures; B, ethanol control; and C r 250 uM

8 -MOP. The ordinate represents the relative number of fluorescent occurrences and the abscissa represents the relative intensity of fluorescence.

65 66 o bi z bi U K (A 111 O 3 -I z bi b. bJ a 0 j c *- o »- 0 © a ® ® N -® h®

I i i i

u ® i—© i— i— i— i— i— r N

o z u LJ O (A O U tt 3 _l b. z bJ bi a (9 j a o *- IS bJ (A bi 3 -I IL AC (9 -I O ►- 0

® SI IS ® * s N ® z ® ® u ® o bJ -* ® -® S O CO CO ® bJ Z

/ f; V, ■ I . £ . i— i— r — t r ‘.'i ■ ■ , — t •./ •./ T— r CD i— r is ® i ® N 002 -MOP -MOP TREATMENT COMPARED TO CONTROLS 8 FIGURE 20. FIGURE 20. THE DNA FLUORESCENCE PATTERN 12 HOURS AFTER FIGURE 21. THE DNA FLUORESCENT PATTERN 36 HOURS AFTER 8-MOP TREATMENT COMPARED TO CONTROLS

~~The cells were treated as in figure 20 except they were analyzd 36 hours after treatment. DNA profile A represents untreated controls; B,~~

~~ethanol controls; and C, 250 uM 8 -MOP treated cultures.~~

~~67 68~~

~~u o z CD ® u - u V I IV o z o o 00 3 u.J ® z ® 111 \0 111 K (0~~

UJ L> ■K z o UJu "V in u) j 9 a ors -|— i— i— r . x fl -® o 3 CM * ® J U. s z II-® UJ J vO UJ tt19 9 J I-® c Ul H u O z *- UJ 9 u r® in *•*» CM 19 UJ c k 3o J U. o i— i— i— i— i— i— i— i— i— r CM ® Z S UJ vO UJ a 19 ® J ♦? I-« I O J H

~~or t— r CM~~

~~FIGURE 21. THE DNA FLUORESCENCE PATTERN 36 HOURS AFTER~~

~~8 -MOP TREATMENT COMPARED TO CONTROLS 69~~

~~22). The increase in cyclic AMP concentration was sustained~~

~~for at least 30 minutes. A 15 minute treatment with~~

~~increasing concentrations of 8 -MOP caused a dose dependent~~

~~rise in cyclic AMP concentration (figure 23). In contrast~~

~~to the fibroblasts, treatment with 8 -MOP altered the cyclic~~

~~GMP concentration in epithelial cells in vitro. After a 15~~

~~minute treatment with 250 uM 8 -MOP, the cyclic GMP~~

~~concentration in epithelial cells was increased over ethanol~~

~~control values and was sustained for at least 30 minutes~~

~~(figure 24). Treatment for 15 minutes with increasing~~

~~concentrations of 8 -MOP did not significantly alter the~~

~~cyclic GMP concentration from that of the ethanol treated~~

~~cells (figure 25). Since the epithelial cell cyclic GMP~~

~~response to 8 -MOP differed from that of fibroblasts, their~~

~~cyclic nucleotide profiles over a 5 day period were~~

~~examined.~~

~~In contrast to the fibroblasts (figure 1), the cyclic AMP~~

~~concentration in epithelial cells decreased with increasing~~

time in culture (figure 26). The epithelial cell cyclic GMP profile was also distinct from that of the fibroblasts. The cyclic GMP concentration at 12 hours after plating was not significantly different from any other determination throughout the 96 hour growth period (figure 27). Although cell counts were not done in this experiment, protein was determined. After the epithelial cells were plated, the protein increased from 270 ug/plate at 12 hours to 784 FIGURE 22. THE TIME DEPENDENT EFFECT OF EFFECT DEPENDENT TIME THE 22. FIGURE CYCLIC AMP (PICOMOLES/MG PROTEIN) 10 12 14 2 4 6 8 0 Dunnett's t-test indicated no difference with difference no indicated t-test Dunnett's IN CELLS EPITHELIAL HUMAN OF CONCENTRATION AMP the zero time control. Analysis by Student's by Analysis control. time zero the difference between ethanol and and ethanol between difference as culture in grown were cells epithelial Human -et niae sgiiat (p<.005) significant a indicated t-test from 5 to 30 minutes. 30 to5 from with treatment minute one significant a and treatment ethanol after time by Analysis AMP. their cyclic forof analyzed then concentration were cells The times. uM 250 with treated and inmethods described CULTURE 8 (p <.005) difference at times longer than the than longer times at difference (p <.005) MP * o ehnl ^ fr h indicated the (^) for (*) ethanol or -MOP 5 10 15 MINUTES 20 8 -MOP when compared to compared when -MOP 8 -MOP ON THE CYCLIC THE ON -MOP 25 8 -MOP treatments -MOP 30

FIGURE 23. THE DOSE DEPENDENT EFFECT OF EFFECT DEPENDENT DOSE THE 23. FIGURE CYCLIC RMP (PICOMOLES/MG PROTEIN) 0 1 12 14 16 2 4 6 6 iue. h el ee hnaaye o their for analyzed then were cells The minutes. M OCNRTO FHMNEIHLA EL IN CELLS EPITHELIAL HUMAN OF CONCENTRATION AMP ocnrtosaoe 25 Mwr significantly were uM 62.5 above concentrations that indicated t-test Student's concentration of cyclic AMP. Analysis by Analysis AMP. cyclic of concentration the with treated and inmethods described as culture in grown were cells epithelial Human CULTURE niae ocnrtos of concentrations indicated p<05 dfeet rmehnl control. ethanol from different (p<.005) 2 5 10 250 100 50 25 0 -EHXPOfLN CUM) 8-METHOXYPSORflLEN 8 MPfr 15 for -MOP 8 -MOP ON THE CYCLIC THE ON -MOP 8 -MOP

««

FIGURE 24, THE TIME DEPENDENT EFFECT OF EFFECT DEPENDENT TIME THE 24, FIGURE CYCLIC GMP (PICOMOLES/MG PROTEIN) .2 .4 .6 .8 0 ramns fe 1 ad 30minutes. and 15 after treatments M OCNRTO FHMNEIHLA EL IN CELLS EPITHELIAL HUMAN OF CONCENTRATION GMP ocnrto vr ie nete the either in time over concentration t-test indicated a significant Student's aby indicated Analysis t-test cultures. treated ethanol significant difference in cyclic GMP cyclic in no was difference There significant GMP. cyclic of concentration as culture in grown were cells epithelial Human CULTURE ie. h el ee hnaaye o their for analyzed then were cells The times. uM 250 with treated and inmethods described 8 (p <.005) difference between between difference (p<.005) MP k o ehnl > fr h indicated the «>) for (k) ethanol or -MOP 5 10 15 MINUTES 20 8 8

~~-MOP and ethanol and -MOP -MOP ON THE CYCLIC THE ON -MOP 530 25~~

~~8 -MOP or -MOP~~

FIGURE 25. THE DOSE DEPENDENT EFFECT OF EFFECT DEPENDENT DOSE THE 25. FIGURE CYCLIC GMP (PICOMOLES/MG PROTEIN) .2 .4 .6 .6 iue. h el ee hnaaye o their for analyzed then were cells The minutes. unt' tts idctdn significant no indicated t-test Dunnett's ocnrtos of concentrations increasing orwith ethanol with treated cultures between concentrations GMP cyclic in difference IN CELLS EPITHELIAL HUMAN OF CONCENTRATION GMP concentration of cyclic GMP. Analysis by Analysis GMP. cyclic of concentration the with treated and inmethods described as culture in grown were cells epithelial Human CULTURE niae ocnrtos of concentrations indicated 2 5 10 250 100 50 25 0 -EHXPOfLN (UM) 8-METHOXYPSORflLEN 8 -MOP. 8 MPfr 15for-MOP 8 -MOP ON THE CYCLIC THE ON -MOP

FIGURE 26. THE EFFECT OF TIME IN CULTURE ON THE CYCLIC AMP CYCLIC THE ON INCULTURE TIME OF EFFECT THE 26. FIGURE CYCLIC AMP (PICOMOLES/MG PROTEIN) 10 12 2 4 6 8 washed twice with PBS and treated with 5% TCA to TCA 5% with treated and PBS with twice washed by radioimmunoassay as described in the methods the in described as radioimmunoassay by section. determined was AMP cyclic of concentration The ua eihla clswr see t4x 10 x4 at seeded were cells epithelial Human CELLS EPITHELIAL HUMAN OF CONCENTRATION C0 cells/60mm plate and incubated at 37° C in a 5%a in C 37° at incubated and plate cells/60mm times, the medium was removed, the plates were plates the removed, was medium the times, inhibit further cyclic nucleotide metabolism. nucleotide cyclic further inhibit i _ 2 4 6 8 0 2 4 96 84 72 60 48 36 24 12 T ______2 ------hmdfe niomn. tte indicated the At environment. humidified 1 ------1 ______1 ------I OR ATR PLATING AFTER HOURS ______1 ------I ______1 ------I ______1 ------I ______1 ------I ______L 1 ------

FIGURE 27. THE EFFECT OF TIME IN CULTURE ON THE ON CULTURE IN TIME OF EFFECT THE 27. FIGURE CYCLIC GMP (PICOMOLES/MG PROTEIN) 2.5 - 1.5 - ocnrto f ylcGPb radioimmunoassay. by GMP cyclic of concentration h el ee rae s ecie i the in described as treated were cells The CELLS CONCENTRATION OF CYCLIC GMP IN HUMAN EPITHELIAL INHUMAN GMP CYCLIC OF CONCENTRATION eedo fgr 2 adaaye o their for analyzed and 26 figure of legend 6 8 0 2 84 72 60 48 36 HOURS AFTER PLATING AFTER HOURS

~~76 ug/plate at 96 hours (figure 28) which is indicative of an increase in cell number.~~

~~Peripheral blood monocytes from normal human donors were~~

also tested with 8 -MOP. Prior to the addition of 8 -MOP, the monocytes were incubated for one hour at 37° C in medium A without FCS to allow their cyclic nucleotide concentrations to stabilize. Immediately after addition of the monocytes into the incubation tubes, the cyclic AMP concentration was

~~29.4 picomoles/mg protein, which decreased to a stable concentration of 12.9 pmoles/mg protein after 75 minutes.~~

~~Upon addition of 250 uM 8 -MOP there was a time dependent increase in the cyclic AMP concentration in the monocytes~~

~~(figure 29). The rise in cyclic AMP concentration was~~

~~similar to that seen in fibroblasts and at 2 0 minutes was~~

~~142% greater than ethanol control levels. Although cyclic~~

~~GMP concentrations were not determined in this experiment,~~

subsequent studies showed that 8 -MOP did not alter the cyclic GMP concentrations relative to control levels during a two hour treatment period (data not shown). In this experiment the cyclic AMP concentrations remained elevated

~~throughout the 2 hour incubation with 250 uM 8 -MOP. In~~

conclusion, 8 -MOP had a similar dose and time dependent effect on cyclic AMP concentrations not only in human fibroblasts, but also in human epithelial cells and monocytes in vitro. FIGURE 28. THE EFFECT OF TIME IN CULTURE ON THE PROTEIN THE ON INCULTURE TIME OF EFFECT THE 28. FIGURE PROTEIN (MICROGRAMS/60 MM PLRTE 200 300 t S00 ora 700 100 ® bj

_i 2 4 6 8 0 2 4 96 84 72 60 48 36 24 12 protein content as described in the methods. the in described as content protein h clswr tetda dsrbd n the in described as treated were cells The eedo fgr 2 adaaye o their for analyzed and 26 figure of legend CELLS EPITHELIAL HUMAN OF CONTENT 1 i ______------i ______OR ATR PLATING AFTER HOURS 1 ------i ______1 ------i ______1 ------i ______1 ------i ______1 ------i ______L r

77 FIGURE 29. THE EFFECT OF EFFECT THE 29. FIGURE CYCLIC AMP (PICOMOLES/MG PROTEIN) 20 25 80 10 15 85 5 AMP concentration between 5 and 20 minutes after 20minutes and 5 between concentration AMP between 5 and 20 minutes (Student's t-test). (Student's 20minutes and 5 between Analysis by Dunnett's t-test indicated a indicated t-test Dunnett's by Analysis ehd adpir o ramn ee incubated were treatment to prior and methods control. Treatment with with Treatment control. ih 5 uM 250 with treatment with with treatment analyzed for their concentration of cyclic AMP. cyclic of concentration their for analyzed ua ooye ee banda dsrbd in described as obtained were monocytes Human significant (p <.005) difference in the cyclic the in difference (p<.005) significant o te niae ie. h clswr then were cells The times. indicated the for treated were cells The C. 37° at hour 1for MONOCYTES BLOOD PERIPHERAL HUMAN IN AMP CYCLIC p <05 dfeet rmehnl treatment ethanol from different (p<.005) 5 8 MP », ae (O) (A) (»), water -MOP , ethanol or 8 8 -MOP ON THE CONCENTRATION OF CONCENTRATION THE ON -MOP 10 -MOP compared to the zero time zero the to compared -MOP MINUTES 8 15 MPws significantly was -MOP 20

~~THE EFFECT OF PSORALEN ANALOGS ON CYCLIC AMP~~

Trimethylpsoralen (TMP) a pharmacologically active psoralen which is used in the treatment of vitiligo (17), was investigated for its effect on the cyclic nucleotide concentration of fibroblasts. TMP was much less soluble in

~~the incubation medium than 8 -MOP. At a concentration of 25 uM, TMP produced a cyclic AMP time response (figure 30)~~

similar to that of 250 uM 8 -MOP (figure 8 ). Both compounds caused an effect within one minute and reached a maximum between 5 and 10 minutes after addition to the culture. The increase in cyclic AMP over control levels was sustained for at least 30 minutes. The effect of a 15 minute incubation with increasing concentrations of TMP is shown in figure 31.

~~The cyclic AMP concentration was increased over control by~~

~~69% at the highest TMP concentration. A concentration of~~

1 .2 % ethanol, used as the vehicle control, did not significantly alter the cyclic AMP level. The cyclic GMP concentration of fibroblasts after treatment did not show a significant change from control (data not shown).

~~These experiments indicated that the pharmacologically active psoralen analog TMP produced a change in the cyclic~~

~~AMP concentration of fibroblasts in vitro which was similar~~

to that seen with 8 -MOP. This effect of TMP and 8 -MOP led to the study of additional psoralen analogs which are being clinically tested for their antipsoriatic effect. FIGURE 30. THE TIME DEPENDENT EFFECT OF TMP ON THE CYCLIC THE ON TMP OF EFFECT DEPENDENT TIME THE 30. FIGURE CYCLIC AMP (PICOMOLES/MG PROTEIN) 0 1 12 14 16 2 8 4 6 minutes. M tetdclswr sgiiaty (pC.005) significantly were cells treated TMP different from ethanol control at 10 and 30 and 10 at control ethanol from different f ylcAP Aayi uigDnets t-test Dunnett's using Analysis AMP. cyclic of M OCNRTO FHMNFBOLSS IN FIBROBLASTS HUMAN OF CONCENTRATION AMP significantly (p <.005) different from the 15the from different (p<.005) significantly el ee hnaaye o ter concentration their for analyzed then were cells TMP uM 25 with treated and inmethods described as culture in grown were fibroblasts Human CULTURE eodteteta l pit fe and after points all at treatment second including one minute. Using Student's t-test, Student's Using minute. one including niae ht the that indicated k o ehnl O fr h idctdtms The times. indicated (O) the for (k) ethanol or 10 15 MINUTES 8 -MOP treated cell were cell treated -MOP 20 25 305

~~0 8~~

FIGURE 31. THE DOSE DEPENDENT EFFECT OF TMP ON THE CYCLIC THE ON TMP OF EFFECT DEPENDENT DOSE THE 31. FIGURE CYCLIC RMP (PICOMOLES/MG PROTEIN) 10 12 2 4 6 8 with TMP are different from the ethanol control ethanol the from different are TMP with AMP CONCENTRATION OF FIBROBLASTS OF CONCENTRATION AMP unt' tts idctdta cls treated cells that indicated t-test Dunnett's as indicated (* p <.01 and ** p <.005). p ** and (*<.01 p indicated as by Analysis AMP. cyclic of concentration their for analyzed then were cells The Human fibroblasts were grown in culture as culture in grown were fibroblasts Human ecie i ehd n rae ih the with treated and methods in described indicated concentrations of TMP for 15 minutes. 15 for TMP of concentrations indicated J — // -i // i i i J— 111 .5 5 25 1 25 10 5 2.5 1 .5 .25 0 t i —ri i— r i i RMTYPOfLN (UM) TRIMETHYLPSORflLEN

~~l i n ______------i ____ 1 ------i __ —i 1— i iii i 1 1 1 --- i i i i i e« 111 ______------i ____ 1 ------~~

~~i i i i —i r i— 1—~~

~~1 8~~

~~8 2~~

5-Methoxypsoralen (5-MOP), 5-methylisopsoralen (5-MIP) and 3-carbethoxypsoralen (3-CEP) (plate 2) were tested for their effect on cyclic AMP concentration in fibroblasts in culture. Due to the limited solubilities of these compounds in 35% ethanol, a maximum of 62.5 uM of 5-MOP and 31.1 uM of

~~5-MIP and 3-CEP were compared to 250 uM 8 -MOP for their effect on cyclic AMP concentrations during a 20 minute incubation period (figure 32). The three analogs did not~~

increase the cyclic AMP concentrations, while 8 -MOP gave its typical time dependent increase. Cyclic GMP concentrations were unaffected by analog treatment (data not shown). The substituents of the psoralen molecule are therefore important for its effects on cyclic AMP. The importance of the integrity of the psoralen backbone on the cyclic nucleotide effect was tested next.

~~Two analogs of 8 -MOP were synthesized by Dr. Dale Sharp at The Ohio State University, Department of Pharmacology~~

~~(plate 3). The 4', 5' double bond on the furan ring was~~

~~hydrogenated (monohydrogenated 8 -MOP) and then the 3, 4 double bond on the coumarin ring was hydrogenated~~

~~(dihydrogenated 8 -MOP), forming molecules which are no longer planar. These analogs were tested for their effect on cyclic AMP concentration (figure 33). In contrast to~~

~~native 8 -MOP, neither hydrogenated analog altered the cyclic~~

~~AMP concentration relative to the ethanol control levels during a 30 minute incubation. OCH~~

~~5-METH0XYPS0RRLEN (5-MOP)~~

~~OCH~~

~~5-METHOXYISOPSORRLEN (5-MIP)~~

~~COOCpHc~~

~~0 ^ > 0~~

~~3-CRRBETHOXYPSORRLEN (3-CEP)~~

~~PLATE 2- THE MOLECULAR STRUCTURE OF 5-METHOXYPSORALEN,~~

3-CARBETHOXYPSORALEN AND 5-METHYLISOPSORALEN FIGURE 32. THE EFFECT OF PSORALEN ANALOGS ON THE CYCLIC AMP CYCLIC THE ON ANALOGS PSORALEN OF EFFECT THE 32. FIGURE CYCLIC AMP (PICOMOLES/MG PROTEIN) 10 2 1 14 16 2 6 8 4 0 Student's t-test indicated no difference between difference no indicated t-test Student's h clswr te nlzdfr their for analyzed then were cells The treatment between 5 and 20minutes. and 5 between treatment significant a but treatments, analog and ethanol concentration of cyclic AMP. Analysis by Analysis AMP. cyclic of concentration irbat ee rw s ecie inmethods described as grown were Fibroblasts n tetdwt ete 20 uM 250 either with treated and CULTURE IN FIBROBLASTS OF CONCENTRATION uM 5-MOP (□), 31.3 uM 5-MIP (A), 31.3 uM 3-CEP uM (A), 31.3 5-MIP uM (□), 31.3 5-MOP uM (p <.005) difference between between difference (p<.005) (v)r or ethanol (O) for the indicated times. indicated the (O) for ethanol (v)r or 5 10 MINUTES 15 8 -MOP and ethanol and -MOP 8 MP (X), 62.5 -MOP 20

~~OCH~~

~~4 1,5'-MONOHYDROGENRTED 8-MOP~~

~~0 OCH~~

~~3,4,4',5'—DIHYDROGENATED 8-MOP~~

~~PLATE 3. THE MOLECULAR STRUCTURE OF MONOHYDROGENATED AND~~

DIHYDROGENATED 8 -METHOXYPSORALEN FIGURE 33. THE EFFECT OF EFFECT THE 33. FIGURE CYCLIC AMP (PICOMOLES/HG PROTEIN) IS 2 1 14 S 2 4 6 8 Analysis by Student's t-test indicated no indicated t-test Student's by Analysis ooyrgntdaao (A) analog , ormonohydrogenated difference between between difference (p<.005) significant a but treatments, analog ewe 25 n 30minutes and 2.5 between AMP. cyclic of concentration their for analyzed difference between ethanol and the hydrogenated the and ethanol between difference ihr h dhdoeae nlg (□), the analog dihydrogenated the either n tetdwt 20 uM 250 with treated and inmethods described as grown were Fibroblasts CULTURE IN FIBROBLASTS OF CONCENTRATION AMP CYCLIC o te niae ie. h clswr then were cells The times. indicated the for s 18 8 -MOP HYDROGENATED ANALOGS ON THE ON ANALOGS HYDROGENATED -MOP 15 MINUTES 8 MPadehnl treatment ethanol and -MOP 825 28 8 MP (X -MOP 1.2% ), and 250 uM of uM 250 and tao (0) ethanol

~~6 8~~

~~THE EFFECT OF ADENYLY CYCLASE RECEPTOR BLOCKERS ON THE 8 -MOP~~

~~MEDIATED INCREASE IN CYCLIC AMP~~

One mechanism by which psoralens might cause a rise in cyclic AMP in cells is through an interacting with receptors that activate the enzyme adenylyl cyclase. In order to test for this possibility, the effect of known adenylyl cyclase receptor agonists and their blockers on cyclic AMP accumulation in fibroblasts in vitro was tested. After a 15 minute incubation with 500 uM isoproterenol, a beta adrenergic agonist, or 500 uM dopamine, a dopaminergic agonist, there was a significant increase in the cyclic AMP concentration of fibroblasts in culture (table 1). A similar incubation with 500 uM histamine or serotonin did not significantly alter the cyclic AMP concentration from control levels. A 15 minute incubation with 500 uM propranolol, an adrenergic blocker, 443 uM haloperidol, a

~~dopaminergic blocker, 500 uM cimetidine, an H2 blocker and~~

500 uM tranylcypromine, a serotonergic blocker, did not significantly alter the cyclic AMP concentration compared to controls. When the cells were pretreated for 15 minutes with these blockers followed by a 15 minute treatment with the appropriate agonist, the increase in cyclic AMP, where demonstrated, was inhibited. These blockers were then

tested for their ability to inhibit the 8 -MOP mediated rise in cyclic AMP (table 2). A 15 minute pretreatment with the TABLE 1. THE EFFECT OF ADENYLYL CYCLASE AGONISTS AND ANTAGONISTS ON THE CONCENTRATION OF CYCLIC AMP IN HUMAN FIBROBLASTS

Fibroblasts were grown as previously described and treated for 15 minutes with 500 uM of the indicated agonist or antagonist or control vehicle. For the combination of agonist and antagonist, the cells were pretreated with 500 uM of the antagonist and then treated for 15 minutes with the matching agonist at a 500 uM concentration- The concentration of cyclic AMP in the cells was determined by radioimmunoassay-

~~8 8 TABLE 1~~

~~THE EFFECT OF AC RECEPTOR AGONISTS/ANTAGONISTS ON CYCLIC AMP~~

~~CONTROL ANTAGONIST AGONIST ANTAGONIST/ AGONIST (picomoles cyclic AMP/ mg protein)~~

~~Isoproterenol 4 5.8+4.1 6.2 + 0.6 5.5 + 0.5 Propranolol 6.3 + 0.6 ______~~

~~Dopamine 29.5 + 6.1 7.6+1.5 7.4+0.6 Haloperidol *6.3 + 0.7 ______~~

~~Histamine 9.5 + 2.1 7.6 + 1.5 9.3 + 0.9 Cimetidine 6.4 + 1.3~~

~~Serotonin 7.1 + 3.0 6.4+0.5 6.6+0.8 Tranylcypromine 7.3 + 0.8 ______TABLE 2. THE EFFECT OF ADENYLYL CYCLASE RECEPTOR BLOCKERS~~

~~ON THE RISE IN CYCLIC AMP INDUCED BY 8 -MOP~~

~~Fibroblasts were grown as described previously and pretreated for 15 minutes with the indicated concentrations of receptor blockers. The cells~~

~~were then treated with 500 uM 8 -MOP for 15 minutes and analyzed for their content of cyclic AMP by radioimmunoassay.~~

~~90 TA B L E 2~~

~~THE EFFECT OF AC RECEPTOR BLOCKERS ON THE 8-MOP MEDIATED RISE IN CYCLIC AMP~~

~~CONTROL ANTAGONIST 8-MOP ANTAGONIST + 8-MOP~~

~~(picomoles cyclic AMP/ mg protein)~~

~~Control 5.5+0-6 11.5+1.1~~

~~5 4 . 5 + 0.3 12.4 + 1.2 Propranolol 50 4 . 7 + 1.0 18.4 + 3.0 500 5.2 + 0.6 14 . 6 + 1.4~~

~~5 5 . 3 + 0.6 16.2 + 1.9 Cimetidine 50 5.7 + 0.2 23.2 + 0.6 500 5 . 7 + 0.7 23.8 + 4.1~~

~~5 6 . 5 + 0.8 25 .8 + 8.1 Haloperidol 50 6 . 4 + 0.3 24 .6 + 4.2 500 5.8 + 0.9 16.0 + 4.7~~

~~Tranylcypromine 500 6.4 + 0.5 7.3 + 0.8 14.2 + 1.7 13.4 + 2.1 various blockers did not inhibit and some cases potentiated the rise in cyclic AMP concentration caused by the~~

~~additional 15 minute treatment with 500 uM 8 -MOP. In one caser propranolol pretreatment was varied between 5 and 15 minutes, which did not effect its inability to block the~~

~~8 -MOP mediated increase in cyclic AMP (data not shown).~~

These results indicated that 8 -MOP did not activate adenylyl cyclase (AC) through the types of receptors tested, since pretreatment with the various blockers did not ihibit the increase in cyclic AMP.

If 8 -MOP were increasing cyclic AMP by activation of AC through a receptor, then the inactive analogs might serve as receptor blockers. The cells were treated for 15 minutes with 62.5 uM 5-MOP, 31.3 uM 5-MIP or 3-CEP or 250 uM of the

~~hydrogenated 8 -MOP analogs and then treated for an~~

~~additional 15 minutes with either ethanol or 250 uM 8 -MOP~~

(tables 3 and 4). Table 2 shows that the results of a 15 minute pretreatment with the analog followed by ethanol treatment did not significantly alter the cyclic AMP concentration relative to controls. However, a 15 minute pretreatment with the various analogs did not inhibit the

~~8 -MOP mediated rise in cyclic AMP concentration. Cyclic AMP was increased over control levels by 55% with ethanol or~~

~~5-MOP pretreatment and approximately 70% with 5-MIP and~~

~~3-CEP. The results shown in table 4, obtained with the hydrogenated analogs as the pretreatment group were not as TABLE 3. THE EFFECT OF PRETREATMENT WITH PSORALEN ANALOGS~~

~~ON THE ABILITY OF 8 -MOP TO INCREASE CYCLIC AMP IN FIBROBLASTS~~

~~Fibroblasts were pretreated for 15 minutes with the indicated concentrations of psoralen analogs or ethanol and then treated for an additional 15~~

~~minutes with either ethanol or 250 uM 8 -MOP. The concentration of cyclic AMP was determined by radioimmunoassay.~~

~~93 TABLE 3~~

~~THE EFFECT OF PSORALEN ANALOGS ON CYCLIC AMP~~

~~PRETREATMENTTREATMENT~~

~~Ethanol 250uM 8-MOP~~

~~(picomoles cyclic AMP/ mg protein) % increase~~

~~Ethanol 5 .48 + 0.15 8.48 + 0 .89 55~~

~~5-MOP (62.5 pM) 5 .59 + 0.15 8.67 + 0.25 55~~

~~5-MIP (31.3 p M ) 5.77 + 0.33 10.15 + 0.91 76~~

~~3-CEP (31.3 p M ) 5 .51 + 0.42 9.46 + 0.29 71~~

~~VO TABLE 4. THE EFFECT OF PRETREATMENT WITH HYDROGENATED~~

~~PSORALEN ANALOGS ON THE ABILITY OF 8 -MOP TO INCREASE CYCLIC AMP IN FIBROBLASTS~~

~~Fibroblasts were pretreated for 15 minutes with the indicated concentrations of psoralen analogs or ethanol and then treated for an additional 15~~

~~minutes with either ethanol or 250 uM 8 -MOP. The concentration of cyclic AMP was determined by radioimmunoassay.~~

~~95 T A B L E 4~~

~~THE EFFECT OF THE HYDROGENATED 8-MOP ANALOGS ON CYCLIC AMP~~

~~PRETREATMENT TREATMENT~~

~~Ethanol 250uM 8-MOP~~

~~(picomoles cyclic AMP/ mg protein) % i n c r e a s e~~

~~Ethanol 7.3+0-8 11.6+1.2 59 mono-hydrogenated 8-MOP (250 pM) 8.9 + 0.6 12.2+0.3 37 di-hydrogenated 8-MOP (250 pM) 8.3 + 0.7 12.9 + 0.6 55~~

~~TMP (25 pM) 13.5 + 2.1 12.5 + 0.9 clear cut for reasons explained below. The monohydrogenated~~

~~8 -MOP analog showed a slight increase in cyclic AMP over~~

~~ethanol control levels which was not seen previously (see~~

~~figure 33). The percent increase in cyclic AMP~~

~~concentration relative to control levels was less~~

~~significant in this experiment because of the slight change~~

~~in the analog-ethanol control cyclic AMP concentration. The~~

~~increase in cyclic AMP mediated by the 15 minute treatment~~

~~with 8 -MOP was not significantly different in any of the~~

~~analog pretreatment groups. Also shown in table 4 is the~~

~~effect of TMP pretreatment on the 8 -MOP mediated change in~~

~~cyclic AMP. The TMP-ethanol treatment caused a significant~~

~~rise in cyclic AMP concentration. The TMP/8 -MOP combined~~

~~treatment was identical to the effect each treatment had by~~

~~itself and, therefore, TMP and 8 -MOP did not cause an~~

~~additive increase in cyclic AMP concentration.~~

~~THE EFFECT OF 8 -MOP ON PROSTAGLANDINS~~

~~Various agents have been shown to increase cyclic AMP~~

~~through stimulation of prostaglandin (PG) biosynthesis, and several PGs have been shown to stimulate an increase in cyclic AMP (84) . Therefore, PGs were measured in the cell~~

~~culture medium after treatment with 8 -MOP to determine whether a change in PG synthesis could explain the increase~~

in cyclic AMP mediated by 8 -MOP. An 8 -MOP time dependent experiment was carried out in which the medium was saved for PG analysis at the time the plates were pulled for cyclic nucleotide analysis. The time dependent increase in cyclic

~~AMP was typical of the 8 -MOP effect (data not shown).~~

~~Analysis of the four PGs, PGE2 , PGF2oc , 6 -keto P G F ^~~

~~(the stable metabolite of prostacyclin) and thromboxane (Tx)~~

~~B 2 (the stable metabolite of TxA2) showed no significant difference in medium concentration over control levels~~

~~throughout the 30 minute treatment with 8 -MOP (figures~~

~~34-37) . In the each figure legend the concentration of the~~

~~PG in the medium prior to addition to the cultures is~~

indicated. Although 8 -MOP treatment does not stimulate the production of any of these PGs, the cells do synthesize these PGs. In an unrelated experiment in which the medium was replaced with fresh medium, the cells were stimulated to synthesize PGs until the concentration of PGs was similar to that in the conditioned medium (data not shown).

~~THE EFFECT OF 8 -MOP ON ADENYLYL CYCLASE~~

The cyclic AMP concentration within a cell is regulated by the activity of two enzymes, adenylyl cyclase (AC) and cyclic AMP phosphodiesterase (PDE). Adenylyl cyclase ++ converts ATP in the presence of Mg into cyclic AMP.

~~Phosphodiesterase converts cyclic AMP to 5 'AMP in the~~

~~_ + + j + + presence of Mg and Ca~~

AC is a membrane bound enzyme which may be linked through a GTP subunit to a receptor. AC activation can occur at any FIGURE 34. THE TIME DEPENDENT EFFECT OF EFFECT DEPENDENT TIME THE 34. FIGURE PGEa CNG/ML) 3 2 s 6 4 1 7 0 ih time. with was no significant difference in PG in difference significant no was hc a aa lvl f .6 gm. There ng/ml. 0.06 of level basal a had which n rae ih 5 uM 250 with treated and inmethods, described as grown were Fibroblasts ocnrto u to due concentration o te niae ie. h eimws then was medium The times. indicated the for CULTURE IN FIBROBLASTS BY PGEj OF PRODUCTION eoe n nlzdfr t cneto PGE of content itsfor analyzed and removed S 10 MINUTES 8 MPo ehnl treatment ethanol or-MOP 20 8 MP X o ehnl (O) (X) ethanol or -MOP 8 -MOP ON THE ON -MOP 2S

~~30IS 2~~

~~100~~

~~1.2~~

~~\ .8 CD~~

~~.8 LU CD 0 _ .4~~

~~0 S 10 IS 20 2S 90 MINUTES~~

~~FIGURE 35. THE TIME DEPENDENT EFFECT OF 8 -MOP ON THE PRODUCTION OF PGF2e( BY FIBROBLASTS IN CULTURE~~

~~Fibroblasts were grown as described in methods,~~

and treated with 250 uM 8 -MOP (x) or ethanol (O) for the indicated times. The medium was then removed and analyzed for its content of PGF2o< which had a basal level of 0.14 ng/ml. There was no significant difference in PG

~~concentration due to 8 -MOP or ethanol treatment with time. FIGURE 36. THE TIME DEPENDENT EFFECT OF EFFECT DEPENDENT TIME THE 36. FIGURE 6 6 KETO PGFlt* (NG/ML) 2.2 2 2.8 2.8~~

.2 A 0 - ih time. with There was no significant difference in PG in difference significant no was There PGFl0<, the stable metabolite of prostacyclin. of PGFl0<,metabolite stable the ocnrto de to due concentration n tetdwt 20 uM 250 with treated and inmethods, described as grown were Fibroblasts CULTURE RDCINO RSAYLNB IRBAT IN FIBROBLASTS BY PROSTACYCLIN OF PRODUCTION eoe n nlzdfr t cnet of content its thenfor was analyzed medium and The removed times. indicated the for S 10 IS MINUTES 8 MPo ehnl treatment ethanol or -MOP 8 20 -MOP (X) or ethanol (X) ethanol or -MOP 8 -MOP ON THE ON -MOP 530 25 6

~~-keto~~

~~101~~

~~(<>)~~

~~1 0 2~~

~~1.8~~

~~1.6~~

~~3Es 49 Z (M CD X *—~~

~~B S 10 IS 20 25 30 MINUTES~~

~~FIGURE 37. THE TIME DEPENDENT EFFECT OF 8 -MOP ON THE PRODUCTION OF THROMBOXANE A? BY FIBROBLASTS IN CULTURE~~

~~Fibroblasts were grown as described in methods,~~

~~and treated with 250 uM 8 -MOP (X) or ethanol (O) for the indicated times. The medium was then removed and analyzed for its content of TxB ,~~

~~(the stable metabolite of TXA2 ) which had a basal level of 0.6 ng/ml. There was no significant difference in PG concentration due~~

~~to 8 -MOP or ethanol treatment with time. 103~~

~~of three locations: the receptor; the guanyl~~

~~nucleotide-binding subunit (G-subunit); or the catalytic~~

~~subunit. Isoproterenol is an agent which increases AC~~

~~activity by activating a specific receptor. The GTP analog~~

~~guanylylimidodiphosphate (GPP(NH)P), which is not hydrolyzed~~

~~to GDP, stimulates AC by activating the G-subunit (6 8 ). NaF~~

~~also appears to act through the G-subunit. A unique~~

diterpene, forskolin, appears to activate adenylyl cyclase by a direct activation of the catalytic site and by indirect activation through potentiation of the modulation of the enzyme by receptors or the guanyl nucleotide-binding subunit

~~(69) .~~

~~8 -MOP was tested for its ability to activate adenylyl cyclase in a cell free system and was compared to the effects of the various types of activators. In figure 38,~~

GPP(NH)P, forskolin and NaF were used as positive controls to show that the fibroblast enzyme preparation was activatable by the agents that worked through the G-subunit and the catalytic subunit. Although these three agents demonstrated a dose dependent activation of the enzyme

~~system, 8 -MOP did not stimulate cyclic AMP production. This~~

~~indicated that 8 -MOP was not activating adenylyl cyclase either through the G-regulatory subunit or the catalytic subunit directly. Another approach to determine whether~~

~~8 -MOP acts through adenylyl cyclase activation was by using~~

2 15'-dideoxyadenosine (DDA) which can inhibit FIGURE 38. THE EFFECT OF EFFECT THE 38. FIGURE CYCLIC AMP (PICOMOLES/MG PROTEIN/MINUTE) 200 250 300 350 400 100 150 - 50- - - .001 mean of 5 samples ± the standard deviation. standard the ± samples 5of mean P(HP (□) (O) GPP(NH)P of f forskolin (A), orconcentrations , indicated NaF the with minutes Membranes were prepared from human fibroblasts human from prepared were Membranes then determined by analysis of cyclic AMP by AMP cyclic of analysis by determined then for incubated and inmethods described as CYCLASE ADENYLYL OF ACTIVITY THE ON ACTIVATORS aiimnasy Ec pit ersns the represents point Each radioimmunoassay. CULTURES FIBROBLAST FROM (x). The activity of the adenylyl'cyclase was adenylyl'cyclase the of activity The (x). DNLL YLS ATVTR (MM) ACTIVATORS CYCLASE ADENYLYL 8 0 . .5 5 25 1 25 10 5 2.5 1 .25 .5.05 .1 -MOP AND KNOWN ADENYLYL CYCLASE ADENYLYL KNOWN AND -MOP 8 -MOP 0 1

~~104 105~~

~~forskolin-activated adenylyl cyclase in intact cells (69).~~

~~A 15 minute pretreatment with 50 uM DDA inhibited the cyclic~~

~~AMP stimulation by 24 and 34% with 10 uM and 50 uM~~

~~forskolin, respectively. However, DDA pretreatment did not~~

~~inhibit the rise in cyclic AMP in cells treated for 15~~

~~minutes with 250 uM 8 -MOP (data not shown). This also~~

~~suggests that 8 -MOP does not activate adenylyl cyclase~~

~~through direct activation of the catalytic subunit.~~

~~Isoproterenol was tested as the positive control to~~

~~determine whether the preparation was activatable by~~

~~receptor linked stimulation. The membrane preparation was~~

~~not activated with isoproteronol treatment alone (results~~

~~not shown). If the isoproterenol receptor linked adenylyl~~

~~cyclase system is intact in the enzyme preparation, then 0 . 1~~

~~uM GPP(NH)P or 1 uM forskolin should potentiate the~~

~~stimulation of adenylyl cyclase with increasing~~

~~concentrations of isoproterenol. As seen in figure 39, the~~

~~enzyme preparation was not stimulated by either~~

~~isoproterenol or 8 -MOP in combination with GPP(NH)P or~~

~~forskolin. From this data, one can not conclude that 8 -MOP does not stimulate adenylyl cyclase through receptor activation, since we were unable to demonstrate intact,~~

receptor-linked catalytic activity. To ensure that the fibroblast cells had a beta adrenergic receptor, cyclic AMP was measured after isoproterenol treatment. The cyclic AMP concentration increased within 15 seconds after addition of IUE3. H EFC FAEYY YLS CIAOS IN ACTIVATORS CYCLASE ADENYLYL OF EFFECT THE 39. FIGURE CYCLIC AMP (PICOMOLES/MG PROTEIN/MINUTE) 200 250 100 150 300 * .5 5 25 1 2 5 10 5 5 250 100 50 25 10 5 2.5 1 .5 .25 1 mean of 5 samples + the standard deviation. standard the + samples 5 of mean ACTIVITY OF ADENYLYL CYCLASE FROM FIBROBLAST FROM CYCLASE ADENYLYL OF ACTIVITY Human fibroblasts were grown in culture as culture in grown were fibroblasts Human determined by analysis of product formation by formation product of analysis by determined (O) 1 or GPP(NH)P uM 0.1 with combination in or preparation was treated forenzyme The treated was preparation inmethods. described as prepared uM forskolin (□). The enzymatic activity was activity enzymatic The (□). forskolin uM aiimnasy Ec on rpeet the represents point Each radioimmunoassay. was enzyme the and analysis enzyme for described CULTURES OR ISOPROTERENOL WITH COMBINATION nraigcnetain o isoproterenol of concentrations increasing dse ie) or (dashedlines) - - SPOEEO O 8MP (UM) 8-MOP OR ISOPROTERENOL 8 MP sldlns aoe (x) alone lines) (solid -MOP 0 1 minutes with minutes 8 -MOP ON THE ON -MOP H > ■

~~106~~

10 uM isoproterenol (figure 40) and by one minute was 13.6 fold greater than control levels. The cyclic AMP concentration declined thereafter with increasing time of exposure to isoproterenol. A 15 minute treatment with increasing doses of isoproterenol caused a dose dependent increase in cyclic AMP (figure 41). Therefore, although the fibroblasts in culture had beta adrenergic receptors which elicited an increase in cyclic AMP when stimulated, the fibroblast enzyme preparations which we used did not retain an intact receptor-linked adenylyl cyclase system which could be stimulated with isoproterenol.

~~THE EFFECT OF 8 -MOP ON PHOSPHODIESTERASE~~

~~Inhibition of phosphodiesterase (PDE), a cytosolic enzyme, causes an increase in cyclic AMP. Two PDE~~

~~inhibitors, papaverine and isobutylmethylxanthine (IBX) were studied for their ability to increase the cyclic AMP~~

~~concentration in fibroblasts in culture compared to 8 -MOP.~~

~~250 uM papaverine did not alter the cyclic AMP concentration throughout a 30 minute incubation period and papaverine did not show a dose dependent response at a 15 minute treatment~~

~~(figures 42 and 43). However, 250 uM IBX caused a rise in cyclic AMP within 2.5 minutes which continued throughout the~~

30 minute incubation (figure 44). Concentrations of IBX up to a maximum of 250 uM caused a dose dependent rise in cyclic AMP concentration (figure 45). The similarities of FIGURE 40. THE TIME DEPENDENT EFFECT OF ISOPROTERENOL ON ISOPROTERENOL OF EFFECT DEPENDENT TIME THE 40. FIGURE CYCLIC AMP (PICOMOLES/MG PROTEIN) 10 0 concentration of cyclic AMP in the cells was cells the in AMP cyclic of concentration the times, indicated the (O) for vehicle (x) drug or isoproterenol uM 10 with incubated and FIBROBLASTS -INOF CONCENTRATION AMP CYCLIC THE eemndb radioimmunoassay. by determined described previously as grown were Fibroblasts CULTURE 15 5 10 MINUTES 20 25 30

8 0 1 FIGURE 41. THE DOSE DEPENDENT EFFECT OF ISOPROTERENOL ON ISOPROTERENOL OF EFFECT DEPENDENT DOSE THE 41. FIGURE CYCLIC RMP (PICOMOLES/MG PROTEIN) J 0 eemndb radioimmunoassay. by determined concentrations indicated the with incubated and described previously as grown were Fibroblasts H YLCAPCNETAINO IRBAT IN FIBROBLASTS OF CONCENTRATION AMP CYCLIC THE concentration of cyclic AMP in the cells was cells the in AMP cyclic of concentration The minutes. 15 for isoproterenol of CULTURE ____ 1. . 1 5 0 0 0 100 50 200 20 10 5 2 1 .5 .1 .2 yf I SPOEEO C 1" M) 10"7 CX ISOPROTERENOL __ I __ I I I I I I I i m u II I m ___ I I L I J.J-1.L1I |

FIGURE 42. THE TIME DEPENDENT EFFECT OF PAPAVERINE ON THE ON PAPAVERINE OF EFFECT DEPENDENT TIME THE 42. FIGURE CYCLIC RMP (PICOMOLES/MG PROTEIN) 2 6 8 4 0 n icbtdwt 20 Mppvrn (x) the or papaverine uM 250 with incubated and described previously as grown were Fibroblasts CULTURE IN INFIBROBLASTS CONCENTRATION AMP CYCLIC concentration of cyclic AMP in the cells was cells the in AMP The cyclic of concentration times. indicated the (O) for vehicle drug between papaverine or control cultures with cultures control or papaverine between time. ifrne n h cci M concentration AMP cyclic no thewas in There difference radioimmunoassay. by determined 5 10 15 MINUTES 20 530 25

FIGURE 43. THE DOSE DEPENDENT EFFECT OF PAPAVERINE ON THE ON PAPAVERINE OF EFFECT DEPENDENT DOSE THE 43. FIGURE CYCLIC AMP (PICOMOLES/MG PROTEIN) 2 4 6 8 and incubated with the indicated concentrations indicated the with incubated and of cyclic AMP in the cells was determined by determined was cells the in AMP cyclic of fppvrn fr 5mnts Te concentration The minutes. 15 for papaverine of described previously as grown were Fibroblasts CULTURE IN FIBROBLASTS OF CONCENTRATION AMP CYCLIC radioimmunoassay. 2 5 10 250 100 50 25 0 AAEIE CUM) PAPAVERINE

~~111~~

'// FIGURE 44. THE TIME DEPENDENT EFFECT OF EFFECT DEPENDENT TIME THE 44. FIGURE CYCLIC AMP (PICOMOLES/MG PROTEIN) 0 1 12 14 16 18 2 6 8 4 0 minutes. the from different (p<.005) significantly were Analysis by Dunnett's t-test indicated that indicated t-test Dunnett's by Analysis CONCENTRATION OF FIBROBLASTS IN CULTURE IN FIBROBLASTS OF CONCENTRATION -et IX rae el ee infcnl (p significantly were cells treated Student's the IBX t-testf Using treatment. second 15 30minutes to 2.5 for IBX with treated cells n icbtdwt 20 M isobutylmethylxanthine uM 250 with incubated and described previously as grown were Fibroblasts ie. h ocnrto f ylcAP n the radioimmunoassay. in by AMP determined cyclic wasof cells concentration The times. .0) rmehnlcnrl ewe 5 n 30 5 and between control ethanol from <.005) ISOBUTYLMETHYLXANTHINE ON THE CYCLIC AMP CYCLIC THE ON ISOBUTYLMETHYLXANTHINE x o te rgvhce O fr h indicated the (O) for vehicle drug the(x) or 5 10 15 MINUTES 20 25 30

~~2 1 1~~

FIGURE 45. THE DOSE DEPENDENT EFFECT OF EFFECT DEPENDENT DOSE THE 45. FIGURE CYCLIC BMP (PICOMOLES/MG PROTEIN) 10 2 4 - 6 - - - OCNRTO FFBOLSS INCULTURE FIBROBLASTS OF CONCENTRATION unt' tts idctdta increasing that indicated t-test Dunnett's and incubated with the indicated concentrations indicated the with incubated and ocnrtos f B ee ifrn from different IBXwere of concentrations Fibroblasts were grown as previously described previously as grown were Fibroblasts tao cnrl s indicated. as control ethanol by Analysis radioimmunoassay. by determined was cells the in AMP cyclic of concentration The minutes. 15 for isobutylmethylxanthine of ISOBUTYLMETHYLXANTHINE ON THE CYCLIC AMP CYCLIC THE ON ISOBUTYLMETHYLXANTHINE 2 5 100 50 25 0 SBTLEHLBTIE C ) M U ISOBUTYLMETHYLXBNTHINE 1 T ----- 1 ----- —I I I I I I 1— J T _____ ----- «* I 1 ___ --- I I 1 -- 250 1

~~__ --~~

~~—I IT I 1— _ I I.I~~

~~113~~

~~114~~

~~the IBX and 8 -MOP cyclic AMP responses suggest that their mechanism of action may be the same.~~

~~In order to test this directly, phosphodiesterase was~~

~~prepared from the fibroblast cultures and the enzyme~~

~~kinetics were analyzed. Double reciprocal plots were used~~

~~to determine the km of PDE (figure 46). A high km~~

~~enzyme and a low km enzyme were observed. To determine~~

~~the k^ of 8 -MOP, increasing concentrations of 8 -MOP were~~

~~added to tubes containing four substrate concentrations.~~

~~Studies using two enzyme concentrations gave similar~~

~~results. At the higher substrate concentrations of 31.3 and~~

~~125 uM cyclic AMP, 8 -MOP did not inhibit PDE (figure 47).~~

~~At 2 and 7.8 uM cyclic AMP, increasing concentrations of~~

~~8 -MOP inhibited PDE-catalyzed metabolism of cyclic AMP. The~~

~~point of intersection defines the of 8 -MOP as approximately 65 uM. 115~~

~~4.5~~

~~3.5~~

~~> \ 2.5~~

~~-.075 -.05 -.025 0 .025 .05 .075 1/S~~

~~FIGURE 46. THE K OF CYCLIC AMP PHOSPHODIESTERASE OF HUMAN^FIBROBLASTS~~

~~The 100,000 X g soluble fraction of a homogenate of human fibroblasts was obtained as described~~

in methods and incubated for 1 0 minutes with several different concentrations of cyclic AMP. The activity of the enzyme was expressed as picomoles 5'AMP formed/mg protein/minute, and the reciprocal of the activity was plotted against the reciprocal of the substrate concentration. 116

~~UJ~ 3.5 t—~~

~~\ Q_~~

~~O t—« _J O >- CJ to UJ _) 7.8 O s: o CJ 31.3 Q_~~

~~> 12S \ - 1 0 0 -50 0 50 100~~

~~8-MOP CUM)~~

~~FIGURE 47. THE K. OF 8 -MOP FOR THE CYCLIC AMP PHOSPHODIESTERASE OF HUMAN FIBROBLASTS~~

~~Phosphodiesterase was obtained from fibroblasts as described in figure 46. The enzyme was incubated with the indicated concentrations of~~

cyclic AMP and 8 -MOP for 10 minutes, and the activity of the enzyme was determined as described in methods. The results were plotted according to the method of Dixon (70 ), and the K. value was calculated by the point of intersection of the lines. DISCUSSION

~~THE BIOLOGICAL EFFECTS OF ULTRAVIOLET LIGHT~~

Ultraviolet light (UVL) causes biochemical, metabolic and structural changes within the cells of the epidermis, the dermis and the immune system. The resulting physiological alterations can be either benefical or harmful. On the positive side, UVL has been used in the treatment of acne for the following reasons. It has bactericidal properties, increases blood flow (which can facilitate toxic product removal) and causes tanning, a cosmetic effect which reduces the visibility of acne-induced inflammation (71) . In addition, UVL interacts with 7-dehydrocholesterol, an endogenous compound in the skin, to form cholecalciferol

(vitamin D^), which is further metabolized to its active form (72) . UVL is also used alone and with psoralens in the treatment of vitiligo and also alone or in combination with coal tar or psoralens for the treatment of psoriasis.

There are a number of less desirable effects of UVL as well. A frequent side effect of first time exposure to UVL of a sufficient intensity is erythema (73) . Upon repeated exposures of UVL, there are protective changes, including tanning and thickening of the epidermis (71) . UVL also causes changes in the skin which are characteristic of

~~117 118 aging. Interaction of UVL with endogenous and exogenous biochemicals in the body can produce phototoxic reactions.~~

~~For example, when porphyrins are inadequately metabolized, their concentration increases. Upon subsequent activation with UVL they can cause massive damage to surrounding tissue~~

(74). Phototoxicity is also a side effect of certain exogenous compounds, such as phenothiazines, anthracene, acridine and some antibiotics (75). Finally, UVL has been implicated as a major factor in the pathogenesis of basal and squamous cell carcinomas and of melanomas (76).

~~The molecular basis for the UVL induced changes seen in cell metabolism and structure are are not well understood.~~

~~Most of the changes have been attributed to photochemical alterations of macromolecules, the most noteworthy being~~

DNA, and to changes in the membrane involving the prostaglandins (PGs). UVL irradiation induces thymidine dimer formation in DNA (77) which interferes with normal DNA function. There is a strong correlation between the lack of repair of this lesion in xeroderma pigmentosum (XP) patients and the numerous malignancies which occur (78). Other

~~UVL-DNA interactions include DNA-protein cross-links in vivo and in vitro (75) and UVL induced single- and double-strand breakage of DNA (79).~~

UVL also increases the prostaglandin-like activity in human skin (80) and may be associated with the pathogenesis of UV induced damage. The inhibitor of prostaglandin 119 synthetase, indomethacin, delays or decreases UV-induced erythema (81,82) and inhibits the UV induced PG increase

(82). Since prostaglandins have numerous biochemical effects (83), they could mediate a variety of changes in epithelial, dermal and immune cells and melanocytes. One important biochemical effect of the prostaglandins is their ability to alter the metabolism of cyclic nucleotides (84).

~~Cyclic nucleotides are important intracellular modulators of proliferation (85,86), maturation (87), intermediate metabolism and cell function in general (37).~~

~~The effect of UVC light on the cyclic nucleotide concentration of human fibroblasts~~

Our initial studies examined the effect of UVC on the cyclic nucleotide concentration of normal human fibroblasts in vitro. Ultraviolet irradiation caused a dose and time dependent increase in cyclic AMP concentration, but did not affect the cyclic GMP concentration. This response was due solely to the exposure of the cells to UV light, since preincubation with the UV screening agent PABA inhibited the effect. The length of time that cells were grown in culture changed their cyclic AMP response to UV light. UVC caused an increase in cyclic AMP concentration in confluent cultures, but caused no change in cyclic AMP in nonconfluent cultures. If this differential response were due to some factor in the medium which was either produced or used by the cells, a change in the medium would alter the response.

In fact, the increase in cyclic AMP concentration due to UV light was independent of the nutritional quality of the culture medium. Therefore, the UV mediated increase in cyclic AMP concentration of fibroblasts in vitro was responsive to the degree of confluency of the cells in culture. Other workers have also shown a difference in the cytotoxicity of UV light depending on the stage of the cell

~~cycle of the irradiated cells (8 8 ) . Although there is no clear explanation for this phenomenon, most cells in confluent cultures are in the same phase of the cell cycle~~

~~(Gq or G^) and also exhibit a high cyclic AMP to cyclic~~

~~GMP ratio.~~

Our studies were done with UVC (150-290 nm) . wavelengths which are rarely, if ever, seen by cells outside of the laboratory. However, other UV wavelengths may also effect the cyclic nucleotide system of cells. Investigation into the cyclic nucleotide response to UVA and UVB would seem appropriate in light of our findings with UVC. This is especially true since wavelengths of 280-315 nm (UVB) cause many of the photobiological reactions that occur in skin such as DNA damage, sunburn and vitamin D production.

Although wavelengths of 315-400 nm (UVA) cause few direct changes, they are responsible for many of the reactions caused by exogenous photosensitizers which cause phototoxic and photoallergic phenomena. 1 2 1

~~The in vivo effects of PUVA~~

~~PUVA is the acronym for the combined treatment of~~

~~8 -methoxypsoralen (8 -MOP) followed by UVA irradiation. It~~

~~is used in the treatment of vitiligo (17) and psoriasis~~

(16). The mechanism by which this treatment causes repigmentation of the albinic patches in vitiligo is unknown. The therapeutic effect of PUVA in psoriasis is probably due, at least in part, to its ability to damage the

~~DNA of epithelial cells (21). In the presence of UVA light,~~

the 3-4 double bond of furocoumarins, such as 8 -MOP, become linked to the pyrimidine bases of DNA (19). The subsequent cross-linking of the adjacent DNA strands through these pyrimidine bases (89) has been shown to inhibit DNA synthesis and cell proliferation in cultured mammalian cells

~~(20). In fact, PUVA has been shown to cause chromosomal damage in a variety of systems (21,90,91.92). This type of~~

DNA damage in the psoriatic epithelial cells could cause the decrease in cellular proliferation seen in the clearing of the lesion, although the effects of PUVA also extend beyond the time necessary for the DNA damage to be repaired (28).

However, not all therapeutic or side effects of PUVA can be explained by DNA damage and the subsequent decreases in cell proliferation. For example, preliminary signs of psoriatic clearing (normalization of capillary loops (29) and return of the stratum granulosum (30)) occur before the 1 2 2 experimental evidence for decreased epithelial proliferation can be detected. In addition, a variety of evidence from immunological studies also points to effects not solely due to UV-psoralen induced DNA damage. For example, treatment

~~of lymphocytes with either 8 -MOP or UVA alone decreased DNA synthesis, and in combination, the effect was additive at lower doses and synergistic at the highest concentration of~~

~~8 -MOP (23) . In vivo treatment of peripheral blood~~

~~lymphocytes (PBL) with 8 -MOP reduced concanavalin A stimulated blastogenesis, a response which is increased in psoriatic cells. Additional treatment of these cells with~~

~~UVA did not decrease the response further (93) , indicating~~

that 8 -MOP had an effect without UV irradiation. There are also humoral immunological changes which do not appear to be directly related to DNA damage. For example, circulating immune complexes were elevated after PUVA (94), though in a massive clinical study there was no evidence that PUVA resulted in a significantly increased number of positive tests for anti-nuclear antibodies (95) . Chemotactic activity in PBL was decreased after in vivo PUVA treatment and also after in vitro treatment of the psoriatic scales or the extracted psoriatic leukotactic factor (32).

~~The mechanism by which PUVA enhances tanning is unknown.~~

Single exposure to UVB and UVA alone or UVA plus 8 -MOP leads to changes in the functional activity of melanocytes, while multiple exposures not only caused functional changes, but 123 also caused an increase in the number of melanosomes (33).

TMP used in combination with ultraviolet light for the treatment of vitiligo was found to increase tyrosinase activity in murine melanoma cells (35) . These authors suggested that UV + TMP induced DNA crosslinks which might

~~block the cells in the G 2 phase of the cell cycle. Cells~~

~~in this phase of the cell cycle have more melanotropin~~

~~receptors available, which could explain their increased~~

~~tyrosinase activity.~~

~~EFFECTS OF PSORALENS~~

~~Our results demonstrate that in the absence of UVL, 8 -MOP and TMP cause a dose and time dependent increase in cyclic~~

~~AMP in normal human fibroblasts in vitro, while cyclic GMP levels are unaffected. TMP is also 10 fold more potent than~~

~~8 -MOP in causing the effect on cyclic AMP- In order to~~

~~determine whether the effect of 8 -MOP was independent of UVL interaction, we pretreated the fibroblast cultures with increasing concentrations of the sunscreen, PABA, followed~~

by 8 -MOP. 8 -MOP caused a rise in cyclic AMP which was not altered by PABA pretreatment even at the highest concentrations tested. At these concentrations, PABA clearly inhibits the UVC mediated increase in cyclic AMP in fibroblast cultures. Although PABA absorbs UV light below

295 nm wavelengths, this experiment did not eliminate the possibility that extraneous UV at longer wavelengths photoactivated the psoralen molecule. Experiments conducted under red light ensured that the dose and time dependent increase in cyclic AMP concentration of fibroblasts was due to the unexcited psoralen molecule.

~~The lowest 8 -MOP dose employed in which the cyclic AMP concentration of fibroblasts was significantly increased above control levels is approximately 10-30 times higher~~

~~then the 8 -MOP serum concentration seen in psoriatic patients (96) . A correlation between serum and epidermal~~

~~8 -MOP concentrations has been demonstrated in the guinea pig~~

(97). However, it is difficult to relate the significance of the phototoxic drug concentrations observed in serum and epidermis to the non-photoactivated drug concentrations which have an effect on the cyclic AMP concentration of fibroblasts in culture.

~~Since the UV mediated effect on the increase of cyclic~~

~~AMP was dependent on the state of confluency of the culture,~~

~~the effect of 8 -MOP on cyclic AMP changes were examined using cultures in different stages of growth. In contrast~~

~~to UV light, 8 -MOP caused an increase in cyclic AMP independent of the state of confluency of the culture. This may be indicative of differences in the mechanism by which~~

~~these two treatments cause a rise in cyclic AMP. The 8 -MOP mediated effect is immediate and probably independent of cell-cell interactions. On the other hand, the UVC mediated~~

~~effect develops slowly, reaching a maximum in 6 hours. It 125~~

~~is demonstrable only in confluent cultures. Therefore,~~

~~intracellular communication may contribute to the mechanism by which UVC irradiation causes an increase in the cyclic~~

~~AMP concentration of fibroblasts. The fact that 8 -MOP has an effect in cultures at different confluencies was~~

~~important for the subsequent experiments on the effect of~~

~~long-term exposure to 8 -MOP.~~

~~Several parameters were measured in the preliminary study~~

~~of the long-term effects of 8 -MOP on cells in culture. One of the parameters examined was cytotoxicity. The number of~~

cells present in the cultures after treatment with 8 -MOP or ethanol was less than the number of cells in the untreated cultures. This decrease did not appear to be due to cell death, since the cells were capable of excluding trypan blue, and no loss of cells was detected by microscopic surveillance for cellular remains. These results indicate that the increasing cell number seen in untreated cultures

~~was inhibited in 8 -MOP and ethanol treated cultures because they decreased cellular proliferation rather than increasing cell death. Pohl and Christophers (98) also showed that, in~~

the absence of UV irradiation, 8 -MOP at concentrations similar to those that we tested was sublethal in guinea pig skin fibroblast cultures as determined by tritiated thymidine uptake and number of adherent cells. Our results do not demonstrate a specific inhibition of cellular

proliferation by 8 -MOP, since a similar inhibition was seen 126 in the control cultures treated with ethanol. Similar experiments must be conducted with a drug vehicle which does not have this inhibitory effect on cellular proliferation to

~~determine whether 8 -MOP has an anti-proliferative effect.~~

~~A second parameter examined in this preliminary~~

~~experiment on the effect of longterm treatment with 8 -MOP~~

was cellular protein. The 8 -MOP treated cultures accumulated less protein/plate throughout the incubation time then either untreated or ethanol treated cultures. The protein/cell in untreated cultures steadily decreased with time in culture, possibly due to the increased density of cells and subsequent decrease in area occupied per cell.

~~Protein/cell in the ethanol treated cultures decreased in~~

~~the first 1 2 hours and thereafter oscillated up and down at~~

24 hour intervals. One explanation for this pattern is that there may be a synchronization of the cell cycle along with an increase in the length of the cell cycle from 12 to 24 hours. Throughout this treatment period the amount of protein/cell remained essentially the same, possibly indicating that there was no restriction due to area

~~limitations in the culture vessel. 8 -MOP treated cells~~

showed an increase in the protein/cell in the first 1 2 hours after treatment, indicating a possible synchronization of the cell cycle and a block in cell division with a concomitant continuation of protein synthesis. Thereafter, at 12 hour intervals the protein/cell oscillated until 48 127

~~hours after treatment, when the protein/cell continued to~~

~~decrease to the level of the untreated cultures. This~~

~~change in pattern also followed the change in the DNA~~

~~fluorescent pattern (figure 2 1 c), which indicated a release~~

~~of the cells from a cell cycle block.~~

~~The cyclic nucleotide measurements in this experiment~~

~~reconfirmed the increase in cyclic AMP and decrease in~~

~~cyclic GMP concentration of fibroblasts with time in~~

~~culture. 8 -MOP caused a typical increase in cyclic AMP~~

~~which was sustained for at least 6 hours. In this~~

~~experiment, ethanol had a slight effect on the cyclic AMP~~

~~concentration which had not been seen previously.~~

~~Suprisingly, the cyclic GMP concentration was higher in~~

~~8 -MOP treated cells than controls, but this did not abolish~~

~~the increased cyclic AMP to cyclic GMP ratio seen after~~

~~treatment. The cyclic AMP/GMP ratio is postulated to~~

indicate the proliferative capacity or state of proliferation in fibroblasts (65,66). The untreated cultures show a continual increase in this ratio except for an unexplained dip at 96 hours after plating. The cyclic

~~AMP/GMP ratio following ethanol treatment remains constant for 24 hours and then increases in parallel to the ratio of~~

~~the untreated cultures. Treatment with 8 -MOP causes the previously seen increase in the cyclic AMP/GMP ratio in the~~

first 6 hours after treatment followed by a return to control levels by 12 hours. It then remains constant. The 1 2 8 immediate increase in the ratio following treatment may signal a halt in cellular replication, and the low cyclic

AMP/GMP ratio in the ethanol and 8 -MOP treated cultures after the initial post-treatment spike is indicative of cultures that are in the lag phase. This is similar to the lag seen in untreated cells just after plating.

~~EFFECTS OF 8 -MOP ON OTHER CELL TYPES~~

The role of fibroblasts in the development of the psoriatic lesion is unknown, and even though the dermis is hyperplastic, fibroblasts have not been extensively studied in relationship to psoriasis. Since epithelial cell hyperplasia is the most obvious result of the disease, we

~~measured the effect of 8 -MOP on the cyclic nucleotide system of normal human epithelial cells in culture. As in the~~

fibroblasts, 8 -MOP caused a time and dose dependent increase in the cyclic AMP concentration of epithelial cells. In contrast to the fibroblasts, the cyclic GMP concentration also increased in a time dependent manner in response to

8 -MOP. Because of this difference, the cyclic AMP and cyclic GMP concentration profiles were determined over a 96 hour growth period. The results obtained from this experiment also contrasted to the data from the fibroblast cultures. Instead of the increase in cyclic AMP seen in fibroblast cultures, epithelial cell cyclic AMP concentrations decreased with time in culture. The cyclic 129

GMP concentration of epithelial cells did not change throughout this time interval. Although cell numbers were not determined in this experiment, an increase in cell number was observed through microscopic observation and can be assumed to be the cause of the increase in protein/plate.

~~However, this is not proof of cellular proliferation, since~~

the epithelial cells have a tendency to differentiate in culture which could account for the increase in protein content. Due to the preliminary nature of these experiments, it is difficult to draw firm conclusions regarding these results.

~~Preliminary experiments using normal peripheral blood~~

~~monocytes demonstrated that 8 -MOP also had an effect on the cyclic AMP concentration of these immunological cells.~~

~~Further studies are needed in order to document the extent~~

of the response of monocytes to 8 -MOP. It also would be interesting to see if this response was indicative of all immunological cell types or, if, as in the case of the epithelial cells and the fibroblasts, there is cell to cell variation.

~~EFFECT OF PSORALEN ANALOGS ON CYCLIC AMP~~

~~As previously mentioned, TMP was 10 times more potent~~

than 8 -MOP in producing the increase in cyclic AMP in fibroblast cultures- Other psoralen analogs were also tested for their potential effect on cyclic AMP. Currently, other investigators are testing a variety of psoralen analogs for their potential use in the treatment of psoriasis (99,100,101). These effects are being correlated to the analogs' ability to cause erythema, monoadducts

~~(99,100) , covalent DNA crosslinking (102) and chromosomal abberrations (103) . We tested 5-methoxypsoralen (5-MOP),~~

5-methylisopsoralen (5-MIP) and 3-carbethoxypsoralen (3-CEP) for their ability to increase the cyclic AMP concentration of normal human fibroblasts in culture. At the concentrations tested, these analogs did not cause a time dependent increase in the cyclic AMP concentration.

Therefore, the substituents on the psoralen backbone are important in the molecules' ability to affect the cellular cyclic AMP concentration. The nature of the psoralen molecule was altered by mono and dihydrogenation of the

~~4',5' and the 3,4 double bonds. Neither of these~~

~~hydrogenated 8 -MOP analogs increased the cyclic AMP concentration over ethanol control levels. Therefore, the~~

~~double bonds that were removed from 8 -MOP, and which impart the planar nature to the psoralen molecule, may also be~~

~~important in the ability of 8 -MOP to increase the cyclic AMP concentration of fibroblasts in vitro.~~

~~MECHANISM OF 8 -MOP ACTION ON CYCLIC AMP~~

~~We investigated three potential mechanisms by which 8 -MOP could cause an increase in the cyclic AMP concentration of normal human fibroblasts in culture: adenylyl cyclase~~

~~activation; phoshodiesterase inhibition; and stimulation of prostaglandin synthesis. An increase in the activity of~~

~~adenylyl cyclase, the membrane bound, synthetic enzyme which~~

~~catalyzes the production of cyclic AMP from ATP, occurs by~~

~~the activation of: 1 ) specific receptors; 2 ) the guanyl nucleotide-binding subunit (G-subunit); or 3) the catalytic~~

~~subunit (68,69). Inhibition of phosphodiesterase, the~~

~~cytosolic enzyme which converts cyclic AMP to 5 'AMP, can~~

~~cause a rise in cyclic AMP concentration in cells.~~

~~Increased prostaglandin synthesis could also mediate a rise~~

~~in cyclic AMP. since prostaglandins can stimulate the~~

~~formation of cyclic AMP (84)-~~

~~Activation of adenylyl cyclase can be modulated by agonists (i.e. isoproterenol) which stimulate through the~~

~~AC-linked receptor, GPP(NH)P, which acts on the G-subunit,~~

~~and forskolin or NaF, which act by directly stimulating the~~

~~catalytic subunit. These agents were used as positive~~

controls to which 8 -MOP was compared for its ability to modulate the activity of fibroblast adenylyl cyclase in a cell free system. Although GPP(NH)P, forskolin and NaF produced dose dependent activation of adenylyl cyclase,

8 -MOP did not activate the production of cyclic AMP by the enzyme. Similar enzyme preparations were not stimulated by isoproterenol, the classical beta adrenergic agonist. This indicated that the preparation had lost the receptor-(G-subunit)-catalytic subunit linkage and was unresponsive to an agonist of the receptor linked to adenylyl cyclase. The requirement for GTP for both

~~activation and inhibition by hormones (6 8 ) was then tested.~~

~~Instead of using GTP, we added either 0-1 uM of GPP(NH)P or~~

~~1 uM of forskolin with increasing concentrations of either~~

~~isoproterenol or 8 -MOP in our fibroblast enzyme preparation.~~

~~The quantities of AC activator used have been shown to potentiate the hormonal stimulation of adenylyl cyclase~~

(69)- In our preparation, the isoproterenol receptor was either not present or the method of preparing the enzyme had altered the receptor or its linkage to the catalytic subunit. To reconfirm the presence of the isoproterenol receptor in fibroblasts that had been cultured, isoproterenol dose and time dependent changes in the cyclic

AMP concentration of fibroblasts were demonstrated. This indicated that the fibroblast does contain a membrane receptor linked to adenylyl cyclase which can be activated by isoproterenol. Therefore, even though the fibroblasts have adrenergic receptors, our membrane preparation of these cells did not retain the intact receptor-catalytic subunit linkage.

~~The adenylyl cyclase inhibitor, 2'5' dideoxyadenosine~~

~~(DDA), which can decrease the forskolin activated enzyme in intact cells, was also tested. Although pretreatment with~~

~~DDA did slightly inhibit the cyclic AMP production of 133 forskolin treated cells, DDA had no effect on the cyclic AMP~~

~~production of isoproterenol or 8 -MOP stimulated cells. The dose of DDA used was therefore capable of partially~~

~~inhibiting forskolin stimulation but incapable of inhibiting~~

~~isoproterenol or 8 -MOP stimulation of cyclic AMP production.~~

~~Following these experiments, the possibility remained that~~

~~8 -MOP could be activating adenylyl cyclase in the whole cell through a membrane receptor.~~

~~To test this, fibroblast cultures were treated with known adenylyl cyclase receptor agonists and their respective antagonists and the effects on cyclic AMP were measured. In~~

~~the cases in which the agonists caused an increase in the cyclic AMP concentration of fibroblasts, pretreatment with~~

~~the appropriate antagonist blocked the response.~~

~~Pretreatment with any of the antagonists tested did not~~

~~inhibit and in some cases potentiated the increase in cyclic~~

~~AMP mediated by 8 -MOP. This indicated that 8 -MOP was not causing stimulation of AC through the adrenergic, histaminergic, dopaminergic or serotonergic receptor. The~~

~~mechanism causing the potentiation of the 8 -MOP response by the receptor antagonists is unknown. Since the antagonists bind to the receptor, normally without activation, it is~~

~~possible that the addition of 8 -MOP causes an alteration of the membrane such that the antagonist has some agonist activity.~~

~~Our experiments did not eliminate the possibility that 8 -MOP works through some untested receptor linked to~~

~~adenylyl cyclase. The analogs of 8 -MOP which did not increase cyclic AMP were therefore tested for their ability~~

to block the 8 -MOP effect. If 8 -MOP does bind to a specific receptor, an inactive analog might serve as a receptor antagonist. None of the non cyclic AMP stimulating psoralen analogs, including 5-MOP. 5-MIP, 3-CEP, monohydrogenated

~~8 -MOP and dihydrogenated 8 -MOP, blocked the 8 -MOP mediated~~

increase in cyclic AMP- Therefore, if 8 -MOP were acting through receptor-linked adenylyl cyclase, these analogs were not acting as receptor blockers. Since the dose and time dependent increases in cyclic AMP induced by the receptor agonist isoproterenol were not similar in amplitude or

pattern to those of 8 -MOP, the possibility arose that 8 -MOP might not be working as an adenylyl cyclase activator. It is possible that inhibition of phosphodiesterase (PDE) is responsible for the rise in cyclic AMP in fibroblast

~~cultures induced by 8 -MOP. To test this, dose and time dependent experiments with the known phophodiesterase inhibitors papaverine and isobutylmethylxanthine (IBX) were~~

~~compared to similar experiments with 8 -MOP- Although papaverine did not increase cyclic AMP concentration in fibroblast cultures at the doses or incubation times tested,~~

~~IBX produced dose and time dependent increases in cyclic AMP~~

~~similar to those seen with 8 -MOP. This is indirect evidence that the mechanism may be the same. 135~~

~~Phosphodiesterase was then obtained from the fibroblast cultures and the k m was determined. Two k ms were observed, one for a high substrate concentration and one for~~

~~a low substrate concentration. 8 -MOP inhibited PDE activity with a k^ of 65 uM. The increase in cyclic AMP concentration in fibroblasts may therefore be due to~~

~~inhibition of PDE by 8 -MOP. A k^ study on other PDE inhibitors would give information on the relative potency of~~

~~8 -MOP as a PDE inhibitor. Since calmodulin is an important~~

modulator of PDE activity (104) , its effect on the 8 -MOP inhibition of PDE activity in an enzyme preparation was determined. The results were not conclusive, in part due to the relatively impure preparation of PDE- and in part due to the low level of activation of fibroblast PDE by calmodulin.

~~In both cases, with and without calmodulin, 8 -MOP inhibited~~

~~PDE to approximately the same extent.~~

~~The third mechanism by which 8 -MOP could be increasing the cyclic AMP concentration of fibroblasts in culture is through stimulation of prostaglandin production.~~

~~Measurement of PGE2 , PGDF2^ and the stable metabolites~~

~~of prostacyclin and thromboxane A 2 in the medium of~~

~~fibroblast cultures after 8 -MOP treatment did not demonstrate a change in these PGs levels with time.~~

Although 8 -MOP did not stimulate the production of the PGs measured, there remained the possibility that some other cyclo-oxygenase or lipoxygenase product was being synthesized in response to 8 -MOP. An alternative way to determine if other prostaglandin-like agents were responsible for the increase in cyclic AMP concentration was to use the cyclo-oxygenase synthesis inhibitor 5.8,11.14 eicosotetraynoic acid (ETYA) and the lipoxygenase synthesis

~~ihibitor, indomethacin. If the 8 -MOP effect on cyclic AMP were mediated by a lipoxygenase or cyclo-oxygenase product, then pretreatment with ETYA or indomethacin would inhibit~~

~~the rise in cyclic AMP seen after 8 -MOP treatment. The results from these experiments were inconclusive. Treatment with 100 uM indomethacin did not inhibit the rise in cyclic~~

AMP mediated by 8 -MOP. However, this concentration of indomethacin was not tested for its ability to inhibit arachidonic acid stimulation of cyclic AMP. On the other hand, 500 uM indomethacin did inhibit the increase in cyclic

~~AMP caused by arachidonic acid and also by 8 -MOP.~~

Unfortunately, at this concentration indomethacin has numerous effects besides its ability to inhibit prostaglandin synthesis (105) . 100 uM ETYA was shown to inhibit the increase in cyclic AMP by arachidonic acid, but also caused a slight increase in cyclic AMP by itself.

~~Pretreatment of fibroblast cultures with ETYA did not~~

~~inhibit the increase in cyclic AMP mediated by 8 -MOP, but caused increase in cyclic AMP concentration which was additive. Therefore, the role of prostaglandins in the~~

~~cyclic AMP effect of 8 -MOP remains uncertain. 137~~

~~SIGNIFICANCE~~

~~An altered cyclic nucleotide system in psoriatic epidermal tissue was first hypothesized by Voorhees in 1971~~

(38). Since that time the role of cyclic nucleotides in psoriasis has not been clearly demonstrated. However, involved psoriatic epidermis has decreased responsiveness to epinephrine, a beta adrenergic agonist (44) , and treatment with practolol, a beta antagonist, induces aggravation of psoriasis and produces psoriatiform cutaneous lesions (48) .

Papaverine and Ro 20-1724, phosphodiesterase inhibitors, have been shown to increase the cyclic AMP level of mammalian epidermis (47,106) and psoriatic epidermal slices in yitro (107) and have been shown to improve psoriatic lesions. Treatment with lithium (an adenylyl cyclase inhibitor) for unrelated psychoses caused an extensive increase in psoriatic lesions and inhibited the effect of usual antipsoriatic treatments (108). Recently, it has been found that calmodulin levels are grossly elevated in the psoriatic lesion (109) . It may be concluded from these results that there is a role for the cyclic nucleotides in the initiation, progression and treatment of psoriasis.

~~The results presented in this document demonstrate that~~

8 -MOP and TMP cause a dose and time dependent increase in cyclic AMP in normal human fibroblasts in vitro - while cyclic GMP levels are unaffected. These effects may be of importance in the clearing of psoriatic lesions induced by

~~PUVA. Although psoriatic epidermis has a decreased responsiveness to beta adrenergic receptor linked adenylyl cyclase (44) this does not preclude the possibility that~~

~~8 -MOP acts by increasing the cyclic AMP concentration, since~~

we demonstrated that 8 -MOP does not act through this receptor. Kinetic in viyp studies indicate that psoriatic epidermal basal cells proliferate more rapidly than uninvolved or normal skin and have an increased turnover

~~rate (6 ). This could be due either to a pathologic cell cycle with a decrease in DNA synthesis time (S phase) or to a recruitment of cells that are normally blocked at or~~

~~G 2 (8,9,10). Cyclic nucleotides may alter psoriatic cells by decreasing their turnover rate. There is evidence that~~

~~an increase in cyclic AMP may block cells in G^ or G 2~~

~~(110) - Therefore, if 8 -MOP induces an increase in cyclic~~

~~AMP in psoriatic lesions similar to its effect in~~

~~fibroblasts in vitro. the 8 -MOP mediated increase in cyclic~~

~~AMP could reinstate the G^ and G 2 block found in normal skin. This is supported by the observation that agents which raise cyclic AMP inhibit the proliferation of~~

~~epidermal cells, probably in the G^ phase, (1 1 1 ) , and elevated cyclic AMP levels induce epidermal cells to keratinize (112,113).~~

Further evidence for the involvement of cyclic nucleotides in PUVA treatment is their role in melanocyte and immune function, both of which are altered after PUVA therapy. The involvement of the immune system in psoriasis

(114,115) has led to the examination of immune cell function before and after PUVA treatment (31). Although the significance of altered immune cell function in psoriasis remains unresolved, the effect of psoralens on monocyte cyclic AMP may be used to hypothesize a potential mechanism

by which psoralens act. For example, 8 -MOP has been shown to inhibit lymphocyte proliferation after stimulation with mitogens (93). It is known that mitogens which stimulate proliferation of peripheral blood lymphocytes increase cyclic GMP concentrations (116). In mouse spleen cells, cyclic GMP induces DNA synthesis and cyclic AMP inhibits the stimulatory activity of cyclic GMP and mitogens (117).

~~Therefore, if the time dependent cyclic AMP accumulation in~~

~~monocytes after treatment with 8 -MOP is indicative of the response of other immune cells, this would explain the psoralen mediated decrease in mitogen stimulated lymphocyte proliferation (93) .~~

Psoriatic patients undergoing PUVA therapy show pronounced darkening of the skin, which may be due to an increase in melanogenesis and melanosome number (34) or tyrosinase activity (35) or melanin production (36).

Melanogenesis occurs in both G 1 and G 2 phases of the cell cycle, and is dependent on cyclic AMP in G-^ and the availability of the adenylyl cyclase-linked melanocyte stimulating hormone (MSH) receptor in G 2 (118,119).

~~Psoralens in combination with UVA induce a G 2 blockade in cultured cells (28) . An increase in cyclic AMP concentration in melanocytes may stop these cells in the~~

~~Gj^ and G 2 phase as in other cell types (1 1 0 ) -~~

~~Therefore, the hyperpigmentation of psoriatic patients following PUVA may be due to an increase in cyclic AMP in melanocytes similar to that which we observed in fibroblasts~~

~~after treatment with 8 -MOP in yitm.~~

Our results indicate that 8-MOP and TMP alter the cyclic nucleotide system of human cells in yitxij. This effect may play a role in the antipsoriatic effect of the PUVA treatment for psoriasis, and may explain some of the effects seen in cells of the immune system and melanocytes after treatment with PUVA. BIBLIOGRAPHY

~~1. Research needs in 11 major areas in dermatology. Journal of Investigative Dermatology. 73:402-413 (1979)~~

~~2. Van Scott EJ. Tissue compartments of the skin lesion of psoriasis. Journal of Investigative Dermatology. 59:4-6 (1972)~~

~~3. Braun-Falco 0. The histochemistry of psoriasis. Annals of the New York Academy of Sciences. 73:936-976 (1958)~~

~~4. Watson W, HM Cannf EM Farber and M Lexie Nall. The genetics of psoriasis. Archives of Dermatology. 105:197-207 (1972)~~

5. Brandrup F, N Holm, N Grunnet, K Henningsen and H Hansen. Psoriasis in monozygotic twins: variation in expression in individuals with identical genetic constitution. Acta Dermatovener. 62:229-236 (1982)

~~6 . Weinstein GD and P Frost. Abnormal cellproliferation in psoriasis. Journal of Investigative Dermatology. 50:254-259 (1968)~~

~~7. Van Scott EJ and TM Ekel. Kinetics of hyperplasia in psoriasis. Archives of Dermatology. 88:373-381 (1963)~~

~~8 . Goodwin P, S Hamilton and L Fry. The cell cycle in psoriasis. British Journal of Dermatology. 90:517-524 (1974)~~

~~9. Gelfant S. The cell cycle in psoriasis: a reappraisal. British Journal of Dermatology. 95:577-590 (1976)~~

~~10. Gelfant S. On the existence of non-cycling germinative cells in human epidermis in vivo and cell cycle aspects of psoriasis. Cell and Tissue Kinetics. 15:393- 397 (1982)~~

~~11. Goeckerman WH. Treatment of psoriasis. Northwest Medicine. 24:229 (1925)~~

~~12. Ippen H. Basic questions on toxicology and pharmacology of anthralin. British Journal of Dermatology. 105 (suppl. 20):72-76 (1981)~~

~~141 142 13. Cram DL. Psoriasis: current advances in etiology and treatment. Journal of the American Academy of Dermatology. 4:1-14 (1981)~~

~~14. Fritsch PO, H Honigsmann, E Jaschke and K Wolff. Augmentation of oral methoxsalen-photochemotherapy with an oral retinoic acid derivative. Journal of Investigative Dermatology. 70:178-182 (1978)~~

~~15. Voorhees JJ and CE Orfanos. Oral retinoids. Archives of Dermatology. 117:418-421 (1981)~~

~~16. Parrish JA, TB Fitzpatrick, L Tanenbaum and MA Pathak. Photochemistry of psoriasis with oral methoxypsoralen and longwave ultraviolet light. New England Journal of Medicine. 291:1207-1211 (1974)~~

~~17. Fitzpatrick TB and MA Pathak. Historical aspects of methoxsalen and other furocoumarins. Journal of Investigative Dermatology. 32:229-231 (1959)~~

18. Melski JW, L Tanenbaum, JA Parrish, TB Fitzpatrick and HL Bleich. Oral methoxsalen photochemotherapy for the treatment of psoriasis: a cooperative clinical trial. Journal of Investigative Dermatology. 68:328-335 (1977)

~~19. Musajo L, F Bordin and R Bevilaqua. Photoreactions at 3655 A linking the 3-4 double bond of furocoumarins with pyrimidine bases. Photochemistry and Photobiology. 6:927-931 (1967)~~

20. Baden HP, JM Parrington, JDA Delhanty and MA Pathak. DNA synthesis in normal and xeroderma pigmentosum fibroblasts following treatment with 8 -methoxypsoralen and long wave ultraviolet light. Biochimica et Biophysica Acta. 262:247-255 (1972)

~~21. Walter JF, JJ Voorhees, WH Kelsey and EA Duell. Psoralen plus black light inhibits DNA synthesis. Archives of Dermatology. 107:861-865 (1973)~~

~~22. Ben-Hur E and MM Elkind. Psoralen plus near ultraviolet light inactivation of cultured Chinese hamster cells and its relation to DNA cross-links. Mutation Research. 18:315-324 (1973)~~

23. Kraemer KH, LH Waters, OL Ellingson and RE Tarone. Psoralen plus ultraviolet radiation-induced inhibition of DNA synthesis and viability in human lymphoid cells in vitro. Photochemistry and Photobiology. 30:263-270 (1979) 143 24. Stern RS, LA Thibodeau, RA Kleinerman, JA Parrish and TB Fitzpatrick. Risk of cutaneous carcinoma in patients treated with oral methoxsalen photochemotherapy for psoriasis. New England Journal of Medicine. 300:809-813 (1979)

~~25. Roenigk HH and WA Caro. Skin cancer in the PUVA-48 cooperative study. Journal of the American Academy of Dermatology. 4:319-324 (1981)~~

~~26. Lassus A, T Reunala, J Idanpaa-Heikkila, T Juvakoski and 0 Salo. Puva treatment and skin cancer: a follow- up study. Acta Dermatovener. 61:141-145 (1981)~~

~~27. Back 0, E Hollstrom, S Liden and W Thorburn. Absence~~

~~of cataract ten years after treatment with 8 - methoxypsoralen. Acta Dermatovener. 60:79-80 (1980)~~

~~28. Cohen SR, DM Carter and M Gala. Proliferative response patterns of human fibroblasts after photoinjury with~~

~~4,5 1 ,8 -trimethylpsoralen. Journal of Investigative Dermatology. 76:10-14 (1981)~~

~~29. Braverman IM and J Sibley. Role of the microcirculation in the treatment and pathogenesis of psoriasis. Journal of Investigative Dermatology. 78:12- 17 (1982)~~

~~30. Fry L and RMH McMinn. The action of chemotherapeutic agents in psoriatic epidermis. British Journal of Dermatology. 80:373-383 (1968)~~

~~31. Morison WL, JA Parrish, KJ Bloch and JI Krugler. In vivo effects of PUVA on lymphocyte function. British Journal of Dermatology. 104:405-413 (1981)~~

~~32. Mizuno N, H Enami and K Esaki. Effect of 8 - methoxypsoralen plus UVA on psoriasis leukotactic factor. Journal of Investigative Dermatology. 72:64-66 (1979)~~

33. Pathak MA, K Jimbow, JA Parrish, KH Kaidbey, AL Kligman and TB Fitzpatrick. Effect of UV-A, UV-B, and psoralen on in vivo human melanin pigmentation, in Pigment Cell Vol. 3 (ed. S.N. Claus). Karger, Basel, 291-298 (1976)

34. Jimbow K and T Uesugi. New melanogenesis and photobiological processes in activation and proliferation of precursor melanocytes after UV exposure: Ultrastructural differentiation of precursor melanocytes from langerhans cells. Journal of Investigative Dermatology. 78:108-115 (1982) 144 35. Carter DM, M Pan and JM Varga. Pigment response of melanoma cells to psoralens and light, in Pigment Cell Vol. 4 (ed. V. Riley). Karger, Basel, 329-336 (1979)

~~36. Rosdahl IK and G Swanbeck. Effects of PUVA on the epidermal melanocyte population in psoriatic patients. Acta Dermatovener. 60:21-26 (1980)~~

~~37. Robison GA, RW Butcher and EW Southerland. Cyclic AMP. Annual Review of Biochemistry. 37:149-174 (1968)~~

~~38. Voorhees JJ and EA Duell. Psoriasis as a possible defect of the adenylyl cyclase-cyclic AMP cascade. Archives of Dermatology. 104:352-358 (1971)~~

~~39. Aoyagi T, K Kamigaki and Y Miura. Cyclic AMP level in different layers of psoriatic epidermis. Journal of Dermatology. 7:131-136 (1980)~~

~~40. Adachi K, H Iizuka, KM Halprin and V Levine. Epidermal cyclic AMP is not decreased in psoriasis lesions. Journal of Investigative Dermatology. 74:74-76 (1980)~~

~~41. Marcelo CL and JJ Voorhees. Cyclic nucleotides and the control of psoriatic cell function. Advances in Cyclic Nucleotide Research. 12:129-137 (1980)~~

~~42. Adachi K, T Aoyagi, 0 Nemoto, KM Halprin and V Levine. Epidermal cyclic GMP is increased in psoriasis lesions. Journal of Investigative Dermatology. 76:19- 20 (1981)~~

~~43. Royer E, J Chaintreul, J Meynadier, B Michel, JJ Guilhou and A Crastes de Paulet. Cyclic AMP and cyclic GMP production in normal and psoriatic skin. Dermatologica. 165:533-543 (1982)~~

44. Yoshikawa K, N Mori, K Hadame and S Sakakibara. Differences in response of psoriatic epidermis in cyclic AMP accumulation against certain adenylyl cyclase agonists. Acta Dermatovener. 60:95-98 (1980)

~~45. Cantieri JS, G Graff and ND Goldberg. Cyclic GMP metabolism in psoriasis: Activation of soluble~~

~~epidermal guanylate cyclase by arachidonic acid and 1 2 - hydroxy-5,8,10,14-eicosatetraenoic acid. Journal of Investigative Dermatology. 74:234-237 (1980)~~

~~46. Harkonen M, VK Hopsu-Havu and K Raij. Cyclic adenosine monophosphate, adenyl cyclase and cyclic nucleotide phosphodiesterase in psoriatic epidermis. Acta Dermatovener. 54:13-18 (1974) 145~~

~~47. Stawiski MA, JA Powell, PG Lang, A Schork, EA Duell and JJ Voorhees. Papaverine: Its effects on cyclic AMP in vitro and psoriasis in vivo. Journal of Investigative Dermatology. 64:124-127 (1975)~~

48. Sondergaard j, s Wadskov, HA Jensen and HI Mikkelsen. Aggravation of psoriasis and occurence of psoriasaform cutaneous eruptions induced by practolol (Eraldin). Acta Dermatovener. 56:239-243 (1976)

~~49. Neumann HAM and T van Joost. Adverse reactions of the skin to metoprolol and other beta-adrenoreceptor- blocking gents. Dermatologica (Basel). 162:330-335 (1981)~~

~~50. Skoven I and J Thormann. Lithium compound treatment and psoriasis. Archives of Dermatology. 115:1185-1187 (1979)~~

~~51. Hawley-Nelson P, JE Sullivan, M Rung, H Hennings and SH Yuspa. Optimized conditions for the growth of human epidermal cells in culture. Journal of Investigative Dermatology. 75:176-182 (1980)~~

~~52. Yuspa SH, P Hawley-Nelson, JR Stanley and H Hennings. Epidermal cell culture. Transplantation Proceedings. 12 (suppl• 1) : 114-122 (1980)~~

~~53. Latarjet SR, P Morenne and R Berger. A simple apparatus for determination of the ultraviolet ray output by germicidal lamps. Annales de 1 Institut Pasteur. 85:174-185 (1953)~~

~~54. Lowry OH, NJ Rosebrough, AL Farr and RJ Randall. Protein measurement with the folin phenol reagent. Journal of Biological Chemistry. 193:265-275 (1951)~~

~~55. Boyum A. Separation of leucocytes from blood and bone marrow. Scandinavian Journal of Clinical and Laboratory Investigation. 21:78-89 (1968)~~

~~56. Steiner AL, CW Parker and DM Kipnis. Radioimmunoassay for cyclic nucleotides. I. Preparation of antibodies and iodinated cyclic nucleotides. Journal of Biological Chemistry. 247:1106-1113 (1972)~~

~~57. Harper JF and G Brooker. Femtomole sensitive radioimmunoassay for cyclic AMP and cyclic GMP after~~

2 ' 0 acetylation by acetic anhydride in aqueous solution. Journal of Cyclic Nucleotide Research. 1:207- 218 (1975) 146 58. Miyachi Y, A Mizuchi and K Sato. Preparation of iodinated cyclic GMP derivitives by lactoperoxidase method. Analytical Biochemistry. 77:429-435 (1977)

~~59. O'Dorisio MS, NS Hermina, TM O'Dorisio and SP Balcerzak. Vasoactive intestinal peptide modulation of lymphocyte adenylate cyclase. Journal of Immunology. 127:2551-2554 (1981)~~

* 60. Fertel RH and B Weiss. Measurement of the activity of cyclic nucleotide phosphodiesterases with firefly luciferin-luciferase coupled assay systems. Methods in Enzymology. 57 (M. DeLuca,ed.):94-106 (1978)

61. Fertel R, JZ Yetiv, MA Coleman, RD Schwarz, JE Greenwald and JR Bianchine. Formation of antibodies to prostaglandins in the yolk of chicken eggs. Biochemical and Biophysical Research Communications. 102:1028-1033 (1981)

62. Traganos F, L Staiano-Coico, Z Darzynkiewicz and MR Melamed. Effects of dihydro-S-azacytidine on cell survival and cell cycle progression of cultured mammalian cells. Cancer Research. 41:780-789 (1981)

~~63. Tejwani GA, R Fertel and RH Hart. The effects of ultraviolet irradiation on cyclic nucleotide concentrations in human diploid fibroblasts. Federation Proceedings. 36:688 (1977)~~

~~64. Fertel RH, GA Tejwani, CR Albrightson and RW Hart. The effect of ultraviolet light on the cyclic nucleotide system of human fibroblasts. Photochemistry and Photobiology. 34:275-278 (1981)~~

65. Moens W, A Vakaer and R Kram. Cyclic AMP and cyclic GMP concentrations on serum- and density-restricted fibroblast cultures. Proceedings of the National Academy of Sciences (USA). 72:1063-1067 (1975)

6 6 . Anderson WB, TR Russell, RA Carchman and I Pastan. Interrelationship between adenylate cyclase activity, adenosine 3':5' cyclic monophosphate phosphodiesterase activity, adenosine 3':5' cyclic monophosphate levels and growth of cells in culture. Proceedings of the National Academy of Sciences (USA). 70:3802-3805 (1973)

~~67. Langner A and AM Kligman. Further sunscreen studies of aminobenzoic acid. Archives of Dermatology. 105:851- 855 (1972) 147~~

~~6 8 . Harwood JP, H Low and M Rodbell. Stimulatory and inhibitory effects of guanyl nucleotides on fat cell adenylate cyclase. Journal of Biological Chemistry. 248:6239-6245 (1973)~~

~~69. Seamon KB and JW Daly. Forskolin, cyclic AMP and cellular physiology. Trends in Pharmaceutical Sciences. 4:120-123 (1983)~~

~~70. Dixon M. The Determination of enzyme inhibitor constants. Biochemical Journal. 55:170-171 (1953)~~

~~71. Sunlight and Man (T.B. Fitzpatrick, ed.) University of Tokyo Press. Tokyo (1959)~~

~~72. DeLuca HF and HK Schnoes. Metabolism and mechanism of action of vitamin D. Annual Review of Biochemistry. 45:631-666 (1976)~~

~~73. Warin AP. The ultraviolet erythemas in man. British Journal of Dermatology. 98:473-477 (1978)~~

~~74. Brodthager H. Polymorphous light eruption, in The biologic effects of ultraviolet irradiation ( F. Urbach, ed.) Pergamon Press, Oxford, 1969)~~

~~75. Kornhauser A. Molecular aspects of phototoxicity. Annals of the New York Academy of Sciences. 346:398- 414 (1980)~~

76. Urbach F, JH Epstein and PD Forbes. Ultraviolet carcinogenesis: experimental, global and genetic aspects, in Sunlight and Man (T.B. Fitzpatrick, ed.) University of Tokyo Press. Tokyo, 259-283 (1959)

~~77. Pathak MA, DM Kramer and U Gungerich. Formation of thymine dimers in mammalian skin by ultraviolet radiation in vivo. Photochemistry and Photobiology. 15:177-185 (1972)~~

~~78. Cleaver JE and JE Trosko. Absence of excision of ultraviolet-induced cyclobutane dimers in xeroderma pigmentosum. Photochemistry and Photobiology. 11:547- 550 (1970)~~

79. Zierenberg BE, DM Kramer, MG Geisert and RG Kirste. Effects of sensitized and unsensitized longwave U.V.- irradiation on the solution properties of DNA. Photochemistry and Photobiology. 14:515-520 (1971)

80. Goldyne ME. Prostaglandins and cutaneous inflammation. Journal of Investigative Dermatology. 64:377-385 (1975) 148 81. Snyder DS and WH Eaglstein. Intradermal anti prostaglandin agents and sunburn. Journal of Investigative Dermatology. 62:47-50 (1974)

82. Black AK, MW Greaves, CN Hensby, NA Plummer and AP Warin. The effects of indomethacin on arachidonic acid and prostaglandins E2 and F2a levels in human skin 24 h after u.v.B and u.v.C irradiation. British Journal of Clinical Pharmacology. 6:261-266 (1978)

~~83. Kuehl FA. Prostaglandins, cyclic nucleotides and cell function. Prostaglandins. 5:325-339 (1974)~~

~~84. Samuelsson B, M Goldyne, E Granstrom, M Hamberg, S Hammarstrom and C Malmsten. Prostaglandins and thromboxanes. Annual Review of Biochemistry. 47:997- 1029 (1978)~~

~~85. Pastan IH, GS Johnson and WB Anderson. Role of cyclic nucleotides in growth control. Annual Review of Biochemistry. 44:491-522 (1975)~~

8 6 . Whitfield JF, AL Boynton, JP MacManus, RH Rixon, M Sikorska, B Tsang and PR Walker. The roles of calcium and cyclic AMP in cell proliferation. Annals of the New York Academy of Sciences. 339:216-240 (1980)

~~87. Friedman DL. Role of cyclic nucleotides in cell growth and differentiation. Physiological Reviews. 56:652-708 (1976)~~

~~8 8 . Chan GL and JB Little. Response of plateau-phase mouse embryo fibroblasts to ultra-violet light. International Journal of Radiation Biology. 35:101- 110 (1979)~~

~~89. Song P-S and KJ Tapley. Photochemistry and photobiology of psoralens. Photochemistry and Photobiology. 29:1177-1197 (1979)~~

90. Pohl J and E Christophers. Photoinactivation and recovery in skin fibroblasts after formation of mono- and bifunctional adducts by furocoumarins-plus-UVA. Journal of Investigative Dermatology. 75:306-310 (1980)

91. Bishop SC. DNA repair synthesis in human skin exposed to ultraviolet radiation used in PUVA (psoralen and UV-A) therapy for psoriasis. British Journal of Dermatology. 101:399-405 (1979) 149 92. Ashwood-Smith MJf EL Grant, JA Heddle and GB Friedman. Chromosome damage in Chinese hamster cells sensitized to near-ultraviolet light by psoralen and angelicin. Mutation Research. 43:377-385 (1977)

~~93. Lischka G and E Decker. Dark effect of 8 -~~

~~methoxypsoralen (8 -MOP) on lymphocytes. Archives of Dermatological Research. 261:267-272 (1978)~~

~~94. Guilhou J-J, J Clot, J Meynadier and H Lapinski. Immunological aspects of psoriasis I. Immunoglobulins and anti-IgG factors. British Journal of Dermatology. 94:501-507 (1976)~~

95. Stern RS, WL Morison, LA Thibodeau, RA Kleinerman, JA Parrish, DEJ Geer and TB Fitzpatrick. Antinuclear antibodies and oral methoxsalen photochemotherapy (PUVA) forpsoriasis. Archives of Dermatology. 115:1320-1324 (1979)

~~96. Honigsmann H, E Jaschke, V Nitsche, W Brenner, W~~

Rauschmeier and K Wolff. Serum levels of 8 - methoxypsoralen in two different drug preparations: correlation with photosensitivity and UV-A dose requirements for photochemotherapy. Journal of Investigative Dermatology. 79:233-236 (1982)

~~97. Kornhauser A, WG Warner and ALJ Giles. Psoralen~~

~~phototoxicity: correlation with serum and epidermal 8 - methoxypsoralen and 5-methoxypsoralen in the guinea pig. Science. 217:733-735 (1982)~~

98. Pohl J and E Christophers. Photoinactivation of cultured skin fibroblasts by sublethal doses of 8 - methoxypsoralen and long wave ultraviolet light. Journal of Investigative Dermatology. 71:316-319 (1978)

99. Dubertret L, D Averbeck, F Zajdela, E Bisagni, E Moustacchi, R Touraine and R Latarjet. Photochemotherapy (PUVA) of psoriasis using 3- carbethoxypsoralen, a non-carcinogenic compound in mice. British Journal of Dermatology. 101:379-389 (1978)

100. Bordin F, F Carlassare, F Baccichetti, A Guiotto, P Rodighiero, D Vedaldi and F Dall'Aqua. 4,5'- dimethylangelicin: a new DNA-photobinding monofunctional agent. Photochemistry and Photobiology. 29:1063-1070 (1979) 150 101. Honigsmann H, E Jaschke, F Gschnait, W Brenner, P Fritsch and K Wolff. 5-methoxypsoralen (Bergapten) in photochemotherapy of psoriasis. British Journal of Dermatology. 101:369-378 (1979)

102. Dali'Aqua F, D Vedaldi, S Caffieri, A Guiotto, P Rodighiero, F Baccichetti, F Carlassare and F Bordin. New monofunctional reagents for DNA as possible agents for the photochemotherapy of psoriasis: Derivatives of 4,5'-dimethylangelicin. Journal of Medicinal Chemistry. 24:178-184 (1981)

103. Ashwood-Smith MJ, GA Poulton, M Barker and M Mildenberger. 5-methoxypsoralen, an ingredient in several suntan preparations, has lethal, mutagenic and clastogenic properties. Nature. 285:407-409 (1980)

~~104. Means AR and JR Dedman. Calmodulin-an intracellular calcium receptor. Nature. 285:73-77 (1980)~~

~~105. De Mello MCF, BM Bayer and MA Beaven. Evidence that prostaglandins do not have a role in the cytostatic action of anti-inflammatory drugs. Biochemical Pharmacology. 29:311-318 (1980)~~

106. Stawiski MA, LJ Rusin, TL Burns, GD Weinstein and JJ Voorhees. RO-20-1724: an agent that significantly improves psoriatic lesions in double-blind clinical trials. Journal of Investigative Dermatology. 73:261- 263 (1979)

107. Rusin LJ, EA Duell and JJ Voorhees. Papaverine and RO- 1724 inhibit cyclic nucleotide phosphodiesterase activity and increase cyclic AMP levels in psoriatic epidermis in vitro. Journal of Investigative Dermatology. 71:154-156 (1978)

~~108. Skott AH, H Mobacken and JE Starmark. Exacerbation of psoriasis during lithium treatment. British Jounal of Dermatology. 96:445-448 (1977)~~

~~109. Van der Kerkof PCM and PEJ Van Erp. Calmodulin levels are grossly elevated in psoriatic lesions. British Journal of Dermatology. 108:217-218 (1983)~~

110. Willingham MC, GS Johnson and I Pastan. Control of DNA synthesis and mitosis in 3T3 cells by cyclic AMP. Biochemical and Biophysical Research Communications. 48:743-748 (1972) 151 111. Delescluse C, N Colburn, E Duell and JJ Voorhees. Cyclic AMP elevating drugs inhibit proliferation of keratinizing epidermal cells in tissue culture. Clinical Research. 22:575a (1974)

~~112. Delescluse C, K Fukuyama and WL Epstein. Dibutyryl cyclic AMP-induced differentiation of epidermal cells in tissue culture. Journal of Investigative Dermatology. 66:8-13 (1976)~~

~~113. Marcelo CL. Differential effects of cAMP and cGMP on in vitro epidermal cell growth. Experimental Cell Research. 120:201-210 (1979)~~

~~114. Cormane RH. Immunopathology of psoriasis. Archives of Dermatological Research. 270:201-215 (1981)~~

~~115. Wahba A. Immunological alterations in psoriasis. International Journal of Dermatology. 19:124-129 (1980)~~

116. Hadden JW, EM Hadden, MK Haddox and ND Goldberg. Guanosine 3':5'-cyclic monophosphate: a possible intracellular mediator of mitogenic influences in lymphocytes. Proceedings of the National Academy of Sciences (USA). 69:3024-3027 (1972)

~~117. Diamantstein T and A Ulmer. The antagonistic action of cyclic GMP and cyclic AMP on proliferation of B and T lymphocytes. Immunology. 28:113-119 (1975)~~

~~118. Varga JM. Cell cycle dependence of melanogenesis in murine melanoma cells, in Pigment Cell Vol. 4 (ed. V. Riley). Karger, Basel, 105-112 (1979)~~

~~119. Cohen SR, DE Burkholder, JM Varga, DM Carter and JC Bartholomew. Cell cycle analysis of cultured~~

~~mammalian cells after exposure to 4,5',8 - trimethylpsoralen and long-wave ultraviolet light. Journal of Investigative Dermatology. 76:409-413 (1981)~~