Teratogenesis, Carcinogenesis, and Mutagenesis 10:439-448 (1990)

Sensitivity of Cultured Lymphocytes From Patients With Nevoid Basal Cell Carcinoma Syndrome to Ultraviolet Light and Phytohemagglutinin Stimulation P. Ferraro, L. Celotti, D. Furlan, 1. Pattarello, and A. Peserico Department of Biology ( P E , L.C., D.E, I,/?) and Clinica Dermatologica (A.P), Padova, Italy DNA repair and replication after in vitro UV irradiation were determined in cultured peripheral blood lymphocytes from 6 patients with nevoid basal cell carcinoma syndrome (NBCCS) and from a group of control donors. DNA repair synthesis (UDS) was measured in unstimulated lymphocytes by incubation with 'H-TdR in the presence of hydroxyurea for 3 and 6 h after UV irradiation (6-48 J/m2). DNA replication was measured in PHA-stimulated lymphocytes, UV-irradiated or mock-irradiated, by incubation with 'H-TdR for 24 h. The effect of the mitogen was followed during 5 days after stimulation by determining the incorporation of 'H-TdR, the increase of cell number, and the mitotic index. NBCCS and control lymphocytes showed equal sensitivity to UV light in terms of UDS and reduced response to PH.4. On the contrary, the mitotic index and the number of cells in stimulated cultures wen: significantly lower in the affected subjects. These data suggest an altered progression along the cell cycle, which could be characteristic of stimulated NBCCS lymphocytes. Key words: NBCCS lymphocytes, DNA repair and replication, mitotic index, mitotic phases, in vitro UV irradiation

INTRODUCTION

The nevoid basal cell carcinoma syndrome (NBCCS) is a rare disorder transmitted as an autosomal dominant trait with marked penetrance and variable expression [ I ] . Its clinical manifestations are multiple basal-cell nevi and cysts of jaws along with skeletal anomalies and various combinations of numerous other defects. The molecular basis of such manifestations is still unknown. Patients treated with therapeutic doses of ionizing radiations develop large numbers of basal cell tumors in the irradiated field [2]. That observation together with the fact that the first tumors appear in the body regions most exposed to light such as the facial skin suggests that the Address reprint requests and correspondence to L. Celotti, Department of Biology, via Loredan 10, 35 13 1 Padova, Italy.

0 1990 Wiley-Liss, Inc.

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high incidence of cancer may be associated with defective repair of DNA damages produced by radiations. In cultured lymphocytes or fibroblasts from NBCCS patients, several authors studied the effects of ultraviolet (UV) light and other mutagens o n cell survival, DNA replication and DNA repair with inconclusive results on the sensitivity to UV light. UV-induced unscheduled DNA synthesis (UDS) in NBCCS cells was reported as either slightly reduced [3], unchanged [4], or delayed [ S ] compared to that occurring in normal cells. In 6 NBCCS subjects we determined the UDS induced by UV and the inhibition of cell proliferation in lymphocytes phytohemagglutinin (PHA)-stimulated after irradiation, by measuring daily the incorporation of tritiated thyrnidine, the mitotic index, and the number of cells. While NBCCS cells showed UV sensitivity similar to the controls, they were characterized by a marked alteration in their responsiveness to PHA. MATERIALS AND METHODS Case Reports

Case 1. This patient is a 14 year-old female. The mother (case 4) is affected by NBCCS. A brother died at the age of 10 years of medulloblastoma. The patient presented some nevoid basal-cell carcinomas, jaw cysts, calcified diaphragma sellae (fused clinoids), macrocephaly, genu valgum, and obesity. Case 2. This patient is a 15 year-old male with some nevoid basal-cell carcinomas, multiple jaw cysts, macrocephaly, scoliosis, and calcified falx cerebri. Case 3. This patient is a 36 year-old female. The father died of medulloblastoma. The patient presents multiple nevoid basal-cell carcinomas, jaw cysts, and calcified falx cerebri. Case 4. This patient is a 39 year-old female. A son died at the age of 10 years of medulloblastoma. A 14 year old daughter (case 1) is affected by NBCCS. The patient presents multiple basal-cell carcinomas, jaw cysts, calcified falx cerebri, genu valgum, scoliosis, bifid ribs, epicanthus, obesity, frontal bossing, and palmar and plantar pits. Case 5. This patient is a 50 year-old female with multiple nevoid basal-cell carcinomas, ovarian carcinoma, calcified thyroid masses, ptosis of kidney, and plantar pits. Case 6. This patient is a 68 year-old male. The patient presented multiple nevoid basal-cell carcinomas, jaw cysts, calcified diaphragma sellae (bridged sella), scoliosis, inguinal hernia, and spina bifida occulta. Control Group

The control group consisted of 7 healthy donors not exposed to UV or gamma rays for professional or therapeutic reasons. Age and sex of the controls were matched with those of NBCCS patients. We excluded from the controls all smokers or donors vaccinated during the last year, women using contraceptive pills, and subjects drinking >SO g alcohol or >3 cups of coffee per day. DNA repair synthesis, DNA replication, and cell proliferation were determined on lymphocytes of all subjects, whereas the analysis of mitoses and the determination of mitotic index were performed on 6 control subjects only. Cells

Lymphocytes were isolated from peripheral blood of NHCCS patients and control donors according to the procedure of Boym [6]. The cells were cultured at 1 X lo6

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cellsiml in Eagle’s essential medium supplemented with 20% foetal calf serum (Seromed) in a humidified incubator with 5% C02. UDS Determination One million lymphocytes were irradiated in open Petri dishes (5 cm diameter) by a 254 nm UVS-11 mineral light lamp giving a fluence rate of 3 J/m2/sec. The cells were exposed to different doses of UV light (6, 12, 24, 48 J/m2) and incubated with 10 pCi/mI of tritiated thymidine t3H-TdR; specific activity 40 Ciimmole, Amity PG) for 3 h in the presence of 2.5 mM hydroxyurea (HU; Sigma). The radioactivity incorporated into DNA was measured by liquid-scintillation spectroscopy as previously described [ 7 ] . UDS was expressed as the difkrence between the radioactivity incorporated by irradiated and unirradiated cells. In lyinphocytes exposed to 24 J/m2 of UV light, UDS was assessed also after 6 h of 3H-TdR incubation. All determinations were done in triplicate. Pattern of PHA Stimulation The lymphocyte suspension was inoculated in 96-well microtitre plates (0.2 ml/well) and supplemented with PHA (M form; Seromed) 10 p,l/well. Starting from the 2nd day after PHA addition, DNA replicative synthesis, cell number, and mitotic index were measured daily up to day 5 . DNA replication was measured in quadruplicate wells by 3H-TdR incorporation (6 yCi/ml, specific activity 6.7 Ci/mmole) (during24 h. At the end of labeling, 1 M NaOH was added to each well to reach the final concentration of 0.3 M and the plates were incubated at 37°C for 3 h. After this time 25 and 50 pl aliquots were taken from each well and macromolecular DNA was precipitated with 10% TCA for 30 min on ice, collected on glass fiber filters (Whatman GF/C), and counted in Packard Filter Count Scintillator. Cell number was determined in parallel cultures by counting with a Haemocytometer the viable cells detected by trypanl blue exclusion. Mitotic index was calculated from separate wells. Cells were spread on glass coverslips, flixed with Helly’s solution, and stained with Mayer’s haemalum. More than 1,000 cells were scored for determining the mitotic index and the distribution of mitotic phases. DNA replication, cell number, and mitotic index were measured also in lymphocytes preirradiated with 24 Jim2 UV light.

Statistics The homogeneity of the mitotic index and of the distribution of the mitotic phases in lymphocytes from each individual among days of observation after PHA addition was preliminarly checked by the G test [8] in order to compare, by the same test, the NBCCS group with the control group. To evaluate cell proliferation in each experimental condition, regression lines were fitted to individual values of cell numbers. Comparisons among slopes were made between the two groups of cultured lymphocytes. RESULTS

The values of UV-induced UDS in lymphocytes from control and NBCCS subjects are shown in Figure 1. UDS was rneasured in unstimulated lymphocytes in the

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Fig. I . DNA repair synthesis (UDS) in NBCCS lymphocytes irradiated with U V light. UDS was meawred in control (a,b) and NBCCS (a’,b’)lymphocytes irradiated with 6-48 Jim’ and incubated with ‘H-TdR lor 3 h (a,a’) or irradiated with 24 Jim’ and incubated with ’H-TdR for 3 and 6 h (b,b’).

presence of HU. The radioactivity incorporated by unirradiated cells, due to the residual DNA replication, was very similar in NBCCS and control cultures, indicating that HU was not more toxic to NBCCS cells. Only in one patient the level of UDS was 2 standard deviations at all doses tested (a, a’). below the mean of control values Lymphocytes irradiated with 24 J/m2 were incubated with ’H-TdR for 3 and 6 h in all control subjects DNA radioactivity increased with time, while in NBCCS patients the increase occurred only in 2 subjects (b, b’). The pattern of the stimulation by PHA was followed in control and NBCCS lymphocytes daily from the 2nd to the 5th day after the addition of the mitogen, by measuring the incorporation of ‘H-TdR into DNA, the percentage of mitotic cells, and the cell number. The incorporation of 3H-TdR in lymphocytes from 7 control subjects and 4 NBCCS patients at different times after PHA stimulation is depicted in Figure 2 for non-irrradiated (a, b) and irradiated (a’, b’) cells. The incorporation of ‘H TdR into DNA varied both in control and NBCCS cells. However, in unirradiated lymphocytes of patients the maximum of DNA radioactivity was reached 24 h later than in the healthy subjects. After UV irradiation, the peak of ’H-TdR incorporation shifted from day 3 to day 4 in 3

*

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days +PHA+UV

0

2

3

4

5

0

2

3

4

5

days

Fig. 2. ’H-TdR incorporation into DNA of lymphocytes at different days after PHA addition. Control lymphocytes: non-irradiated (a), UV-irradiated w7ith 24 J/m2 (a’), NBCCS lymphocytes: non-irradiated (b), UV-irradiated (b’).

controls. Given the variability of the individual patterns of 3H-TdR incorporation in both groups of donors, we did not apply any statistical test to compare DNA radioactivity between and within the two groups. The percentage of mitotic cells in cultured lymphocytes from control and NBCCS subjects is reported in Figure 3 . In the controls the mitotic index showed its highest value at day 3 of PHA stimulation, independently of UV irradiation, while in the patients the peak of mitoses occurred 24 h later and was much lower. The maximum values of mitotic index were compared between groups by the G test and were found significantly higher ( P < .001) in healthy than in NBCCS lymphocytes, both UVirradiated and non-irradiated. The irradiation did not cause significant variations of the mitotic index within the two groups. Besides the overall percentage of mitoses, the distribution of the individual mitotic phases was scored at each day in control and NBCCS lymphocytes (Table I). As the distribution of the mitotic phases in each subject was homogeneous among the dif-

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3

2

El x

u 1 C .-

-

U

c 0

c

.E 0

2

3

4

5

0

2

3

5

4

days

tb

J

3

0

2

+PHA+UV

1

3 - h - s "

days

Fig. 3 . Mitotic index during different days of PHA stimulation in control (a,a') and in NBCCS lymphocytes (b,b') non-irradiated (a,b) and UV-irradiated (a',b') with 24 Jim2. Differences between patient and control groups were significant ( P < ,001) at the G test.

ferent days of observation, we chose the day with the highest mitotic index to compare the two groups of lymphocytes. In NBCCS cells the distribution of mitotic phases was significantly different from that in controls ( P < .001), due to an accumulation of anaphases and telophases. The same applied to UV-irradiated NBCCS cells compared to controls (not shown). The number of viable cells in the cultures at each day of stimulation increased according to significant regression lines (Fig. 4). In all cases no significant departure from linearity (residual variance) was detected. The comparison of the slopes showed significant differences between patient and control lymphocytes, both irradiated and non-irradiated ( P < .025 for each comparison). On the contrary, the difference between the two slopes (non-irradiated and irradiated) within patient and control groups was not significant.

1 (rk)

Control subjectsa

Cells scored

6 (L)

5 (4

4 (MI

3 (0)

2 (*)

1,519 1,042 1,358 1,394 1,030 1,031 1,026 1,016 1,067 1,034 1,067 1,050 1,042 1,038 1,032 1,063 1,044 1,062 1,052 1,033 1,037 1,067 1,041 1,053

P 5 3 -

1 -

1 1

-

2 1

I

2 3 1

2 3 1 1

1

1 4 1 2 1 1 2 1 2 5 3

Mitotic phasesh M A 2 13 I 2 4 12 8 3 4 3 3 6 19 16 14 3

-

1

8 9 6 5 12 7 7

-

-

1

-

1

-

3 4 8 6 2

2 3 2

1

3 4

T

-

6 1

5 3 12 9 3 2 3 3 4

Total mitoses 6 26 2 7 8 17 9 6 5 14 ii 6 8 32 21 21 9 23 19 10 7 18 13 13

1(0)

NBCCS subjects"

Cells scored

(a)

6 (V)

5 (D)

4 (0)

3

2 (0)

1,009 1,058 1,070 1,091 1,052 1,013 1,080 1,072 1,053 1,056 !,058 1,064 1,03 1 1,015 1,016 1,025 1,019 1,021 1,049 1,076 1,106 1,054 1,016 1,039

-

-

2

2

Mitotic phases' M A

1 1 1 1

-

1

-

1 3 2 2 !

1

1

1 1 I

-

1

1 3 3

-

-

2 1

1 1

1

1

-

-

-

1

1

I

2 2

I

-

-

-

-

-

1 2 l 3

-

-

-

1 1 1 -

1

-

-

P

TABLE I. Mitotic Index and Distribution of Mitotic Phases in Control and NBCCS Lymphocytes at Different Days of PHA-Stimulation Days of stimulation 2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5 "Symbols inside brackets are those used for the same subjects in Figures 2-4. bP, prophases; M , metaphases; A , anaphases; T, telophases.

T

1 4 3 3 2 2 4 4 2 6 6 5 1 4 5 3 2 2 2 1 3

4 12 8

Total mitoses

6

4 5 7 4 4 6 8 6 3 6 10 8 3 6 9 5 3 4

2 4 1 13 12

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,/

1 0

2

3

4

5

0

,,

,

2

3

4

5

days

Fig. 4. Cell proliferation in control (a,a’) and NBCCS (b,b’) lymphocytes non-irradiated (a,b) and UV-irradiated (a’,b‘) with 24 J/m’ at different days of PHA stimulation. The regression line for a is: y = -0.93 + 0 . 9 1 x , P < . 0 2 5 ; f o r b : y = 0 . 1 4 + 0 . 3 7 x , P < . 0 5 ; f o r a ’ : y = - 0 . 3 6 + 0 . 6 3 x , P < . I l l ; for h‘: y = 0.21 + 0 . 3 2 x , P < .0S. In each case no significant residual variance was observed. Diffcrences between the slopes of the two groups were significant, P < ,025.

DISCUSSION

The studies carried out at the cellular level have not reached a conclusion on the molecular basis of NBCCS . Cytogenetic analysis on lymphocytes stimulated with PHA showed that chromosomal instability is not a specific trait of the syndrome [4,9, lo]. Other authors have examined NBCCS cell ability to repair DNA damage induced by mutagenic treatments. A 25% reduction of UDS in NBCCS lymphocytes was measured by Ringborg et al. [3] after UV irradiation (9.6 and 19.2 J/m2). Nagasawa et al. (51 employed autoradiography to measure the UDS induced by increasing UV doses in NBCCS fibroblast strains, and found that a plateau was reached at 10 or 15 J/m2, i.e., at lower UV doses than in normal fibroblasts. They suggested DNA repair in NBCCS cells to be slower than in controls, given that at 24 h from UV irradiation the incorporation of 3H-TdR was higher in NBCCS than in control fibroblasts. In our experiments on NBCCS lymphocytes the UDS values were within control limits in 4 out of 5 patients. Accordingly, Stefanini et al. [4] did not report significant differences in UDS between lymphocytes from 4 NBCCS patients and controls after 12 J/m2 UV irradiation. Here UDS reached a plateau between 6 and 24 Jim2 in most control subjects

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(Fig. la) while in NBCCS cells a clear plateau was observed only in the patient with very low repair ability (Fig. la’). When ithe incubation with 3H-TdR was prolonged from 3 to 6 h, the incorporation of TdR by lymphocytes of control donors continued, reflecting the persistence of DNA repair at low rate. Only 2 patients gave a similar pattern. Our results indicate that in NBCCS lymphocytes UDS neither is delayed as was observed in fibroblast strains [ 5 ] ,nor reaches a plateau at lower UV doses than in control lymphocytes. The UDS levels detected in cells exposed to UV light or other mutagens are highly variable in human populations [7,11,12]. Therefore, when small groups of individuals are examined, as is the case with NBCCS patients due to the low frequency of the syndrome, the results obtained may not be conclusive. Instead, when the DNA repair deficiency is the major feature of the genetic condition, as in xeroderma pigmentosum, the differences are marked enough to be statistically significant in any case. UDS is the parameter most frequently employed to evaluate DNA repair in cultured cells, given the simple procedure required to measure it. However, many different factors may influence the amount of radioactivity incorporated into DNA, so that UDS may result in an imprecise measure of DNA repair. For these reasons, we examined the sensitivity to UV irradiation in NBCCS cells also by stimulating with PHA the irradiated lymphocytes and measuring their ability to respond to the mitogen, which is the result of UV-cytotoxicity and the repair of the induced damage. NBCCS lymphocytes were rather homogeneous in their response to PHA stimulation, which was lower than in control lymphocytes. The pattern of 3H-TdR incorporation into DNA was rather erratic both in NBCCS and control lymphocytes (Fig. 2), possibly because besides reflecting the actual level of DNA synthesis, it is affected by other factors. The size of the endogenous nucleotide pool and the activity of thymidine kinase, responsible for TdR incorporation into the pool, influence the specific activity of dTTP in cells incubated with 3H-TdR [ 13-15]. Differences in dTTP specific activity in lymphocytes from different donors may prevent a direct comparison of DNA synthesis based on the radioactivity incorporated into DNA. The mitotic activity of NBCCS lymphocytes was significantly different from that of controls: the peak of mitoses was reached 1 day later and was significantly lower. The frequency of the various mitotic phases was also different in NBCCS patients, with an excess of anaphases and telophases. A delay in the peak of mitoses was reported for NBCCS lymphocytes also by Stefanini et al. [4], who, however, did not find any difference in the value of mitotic index between patient and control cells at 100 h of PHA stimulation, when the highest frequency of mitoses in NBCCS lymphocytes was reached. Those authors scored mitoses after 5 h of incubation with Colcemid; therefore, their experimental conditions were diifferent from ours. The overall picture obtained from our data suggests an alteration of the cell cycle progression in NBCCS lymphocytes stimulated with PHA. In NBCCS cell cultures mitosis did not proceed regularly: the increased frequency of anaphases and telophases (Table I) could reflect a difficulty in completing cell division. The reduced number of lymphocytes could result from cell death at late mitosis and/or a lower and delayed responsiveness to PHA . Given the homogeneous behaviour of lymphocyte cultures from different NBCCS patients as regards the alteration of the cell cycle, we are led to think that this represents a specific feature of NBCCS cells, which could have immunological consequences of NBCCS patients, provided that the in vitro observations reflect what takes place in vivo during the immune response.

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ACKNOWLEDGMENTS

We are grateful to Vera Bianchi for helpful comments and suggestions and to Antonella Russo for assistance with the statistical analysis. This work was supported by grants from the National Research Council (CNR) of Italy (PF “Oncologia”), the Italian Association for Research on Cancer (AIRC), and the Venetia Region. REFERENCES I . Gorling RJ: Nevoid basal-cell carcinoma syndrome. Medicine (Baltimore) 66:98-113, 1987. 2. Strong LC: Genetic and environmental interactions. Cancer40: 1861-1866, 1977. 3. Ringborg U, Lambert B, Landegren J , Lewenshon R: Decreased UV-induced DNA repair synthesis in peripheral leukocytes from patients with the nevoid basal cell carcinoma syndrome. J Invest Dermatol 76:268-270, IV81. 4. Stefanini M , Lagoinarsini P, Berardesca E, Borroni G , Rabbiosi G , Nuzzo F: Normal sensitivity to mutagens, spontaneous chromosome breakage, and mutation frequency in nevoid basal cell carcinoma syndrome. Arch Dermatol Res [Suppl] 280: 19-23, 1988. 5 . Nagasawa H. Burke MJ, Little FF, McCone EF, Chan GL, Little JB: Multiple abnormalities in the ultraviolet light response of cultured fibroblasts derived from patients, with the basal cell nevus syndrome. Teratogenesis Carcinog Mutagen 8:25-33, 1988. 6. Boym A: Separation of lymphocytes, lymphocyte subgroups and monocytes: a review. Lymphology 10:71-76, 1977. 7. Celotti L, Furlan D, Ferraro P, Levis AG: DNA repair and replication in lymphocytes from smokers exposed in vitro to UV light. Mutat Res 4:82-86, 1989. 8. Sokal R R , Rolf FJ: “Biometry. The Principles and Practice of Statistics in Biological Research,” 2nd Ed. New York: Freeman WH and Company, 1981. 9. Romke C, Godde-Saltz E, Grote W: Investigations of chromosomal stability in the Gorlin-Goltz syndrome. Arch Dermatol Res 277:370-372, 1985. 10. Sarto F, Mazzotti R, Tomanin R, Corsi GC, Peserico A: No evidence of chromosomal instability in nevoid basal-cell carcinoma syndrome. Mutat Res 225:2 1-26, 1989. 1 1 . Oesch F, Aulmann W, Platt K, Doerjer G: Individual differences in DNA repair capacities in man. ArchToxicol [Suppl] 10:172-179, 1987. 12. Setlow RB: Variations in DNA repair among humans. In Harris CC, Autrup H: “Human Carcinogenesis.” New York: Academic Press, 1983, pp 231-254. 13. Bianchi V, Zantedeschi A, Levis AG: The scintillornetric evaluation of DNA repair synthesis can be distorted by changes of thymidine pool radioactivity. Chem Biol Interact 43:17-3 1, 1983. 14. Spyrou G , Reichard P Dynamics of the thymidine triphosphate pool during the cell cycle of synchronized 3T3 mouse fibroblasts. Mutat Res 200:37-43, 1988. IS. Arner ESJ, Flygar M, Bohman C, Wallstrom B, Eriksson S: Deoxycytidine kinase is constitutively expressed in human lymphocytes: consequence for compartmentation effects, unscheduled DNA synthesis, and viral replication in resting cells. Exp Cell Res 178:335-342, 1988.