Specific Cellular Defects in Patients with Fanconi Anemia ROSANNA WEKSBERG, MANUEL BUCHWALD, ' PATRICIA SARGENT, MARGARET W. THOMPSON AND LOUIS SIMINOVITCH Department of Genetics and Research Institute, Hospital for Sick Children, Toronto, Ontario M5G 1x8 and Department of Medical Genetics, University of Toronto, Toronto, Ontario, Canada

ABSTRACT Measurements of plating efficiency, accumulation of metaphases and generation times have shown that fibroblast from patients with Fanconi anemia (FA) have decreased probability of completing a further division after successful mitosis. Thus FA cells show decreased growth rates and increased generation times. We have also measured the survival of FA fibroblasts and lymphoblasts after treatment with a variety of mutagens. All FA cells show an increased sensitivity to drugs such as MMC and psoralen plus long wave length UV which cause DNA interstrand crosslinks. FA strains show varying degrees of sensitivity to these drugs and the extent of this sensitivity seems to be characteristic of each patient. FA cells are equal t o controls in their sensitivity to other alkylating agents such as ethyl methane sulfonate, Nmethyl-N I-nitro-N-nitrosoguanidine and actinomycin D. Both the decreased growth and increased drug sensitivity may result from defect in DNA replication or repair. Fanconi anemia (FA) is one of a number of genetic disorders known to involve chromosome breakage and a high risk of malignancy (German, '72). Patients with FA exhibit disturbances of organ development and of growth from the earliest period of life. They present with intrauterine growth retardation, short stature, congenital anomalies and bone marrow failure (Fanconi, '67). Chromosome breakage can be demonstrated in lymphocytes, fibroblasts, and bone marrow of these patients (German,'72). Thelow birthweight,short stature, congenital anomalies and chromosomal aberrations all suggest that an impairment of cell division underlies the growth problem in this disorder. Indeed, cells from FA individuals have been reported to grow poorly in culture (Young, '71) and Elmore and Swift ('76) observed that FA fibroblast strains had a mean population doubling time of 30 hours compared with 23 hours for controls. The association of malignancy with a disease of autosomal recessive inheritance such as FA, and the increased incidence of malignancy in carriers of this gene (Swift, '76) indicated that investigation of the defect in FA might provide important information on a t :

J. CELL. PHYSIOL. (1979)101: 311-324.

least one of the mechanisms of neoplastic transformation. While several different pathways of human DNA repair are known to exist, the detailed mechanisms have not yet been described. Past studies with bacteria and human cells have shown that DNA repair disturbances may manifest as increased sensitivity (decreased survival) on exposure to radiation and to various chemical agents (Grossman et al., '75; Setlow, '78). Survival studies are thus a useful first step in screening for specific human DNA repair deficiencies. For example, the demonstration that cells from patients with Xeroderma Pigmentosum (XP) are UV sensitive was the first step in the continuing study of defective repair of UV induced DNA damage in these cells (Cleaver, '69; Setlow, '78). As a primary approach to the examination of the defect in FA patients, we therefore decided to examine two aspects of the behaviour in vitro of cells from such patients, the growth cell parameters and the cell sensitivity to a variety of alkylating and radiomimetric agents. In respect to the former Received Feh. 2, '79.Accepted June 7, '79. ' To whom to address correspondence.

311

312

WEKSBERG, BUCHWALD, SARGENT, THOMPSON AND SIMINOVITCH TABLE 1

Source of Fanconi anemia cell strains Patient

Sex

RS

F

LL

F

Age

8 9.5 8

11 RG

EP MB MC SW CB -

M M F M F F M M

8

10 12 2 10.5 16 20 3

6 3

Fibroblast strain number

Site of biopsy

Lymphoblast line number

Forearm Forearm Forearm Forearm Forearm Forearm Forearm Forearm Forearm Forearm Forearm Forearm

FA-52 FA-182 FA-61 FA-404 FA-67 FA-151 FA-421 FA-145 FA-156 FA-400 FA-402 FA-421 FA-195 FA-198 *



HSC62 HSC72 HSC99

?

Deltoid

‘Obtained from American Type Culture Collection (CCL 122-HG 261) Obtained from Montreal Cell Repository (RS).

problem, the prolonged doubling time of cells from FA patients might mean that (a) a certain fraction of patients’ cells is “dying” a t each division, i.e., is unable to proceed through another divison; or that (b) the generation time of individual cells from patients is longer than that of cells from controls, or both. We present evidence here, using three independent measures, that FA cells have a relatively low division probability. In addition, the decreased growth seen in FA cells can also be a t tributed to an increased generation time (To). To examine the second parameters we tested DNA repair in FA cell strains, beginning with the profile of FA sensitivities to a variety of agents. Some indications of such a defect were provided by the results of Sasaki and Tonomura (‘73) who showed an increased frequency of chromosome breakage in peripheral blood lymphocytes from FA patients after treatment with mitomycin C or with psoralen and long wavelength UV light. The agents we have used include 4 types of alkylating agents: ethylmethane sulfonate (EMS), mitomycin (MMC), N-methyl-”nitro-N-nitrosoguanidine (MNNG), and psoralen plus long wave length UV, as well as actinomycin D, a radiomimetric agent (Bachetti and Whitmore, ’69).We show here that the FA cells exhibit a highly specific sensitivity to MMC and to psoralen plus long wavelength UV light. A preliminary report of this work has been published in abstract form (Finkelberg e t al., ’74). Recent reports have shown that FA fibroblasts are sensitive to MMC

(Fujiwara et al., ’77), psoralen and long wave length UV (Sasaki, ’78) and that lymphocytes are sensitive to MMC when tested with a micronucleus assay (Heddle et al., ’78). MATERIALS AND METHODS

Cell culture Fibroblasts strains were initiated from skin biopsies as described by Goldstein and Littlefield (’69). Control strains were obtained from volunteers and from surgical patients. Fanconi anemia cells were derived mainly from patients diagnosed a t The Hospital for Sick Children (HSC), Toronto, on the basis of clinical and hematologic findings and chromosome breakage studies. Two FA strains were obtained through tissue culture banks. More detailed information regarding the FA patients from HSC may be found in Finkelberg (‘77). All strains were grown in a-medium (Stanners et al., ’71) supplemented with 15%fetal calf serum (Flow Laboratories, Rockville, Maryland). Cells were grown as monolayers in 100-mm plastic Petri dishes (Falcon) in a humidified atmosphere of 5% COz, 95% in air. The culture medium was changed twice weekly unless otherwise indicated. After reaching confluence, cells were subcultured for further propagation using two washes of PBS, trypsinization with 0.25% trypsin (Difco) for 5-10 minutes, and resuspension of cells in a-medium. Every 1:2 transfer (split) was considered 1 generation (i.e., 1 cumula-

CELLULAR DEFECTS IN FANCONI ANEMIA

tive population doubling) and so recorded. After 2 weeks of growth in antibiotic-free medium all strains were routinely tested for mycoplasma, including T-strains, by enrichment culture in 4 media with and without yeast extract, and supplemented with horse and porcine serum. Lymphoblasts lines were started from the same FA patients who donated skin biopsies and from control volunteers using the method of Glade and Broder (’71) (table 1). Cells were cultured a t 37°C in the same medium as the fibroblasts either as clumps in flasks or a single cell suspension in roller tubes or spinner flasks, and counted in an electronic cell counter (Particle Data, Inc.) or in a haemocytometer. Viability was measured by dye exclusion and only cultures with greater than 95% viability were used for experiments. Lymphoblast lines were free from mycoplasma infection when tested as described above.

313

agarose (Sea Plaque Agarose, Marine Colloids Inc., Rockland, Maine). Lymphoblasts were then suspended in 2.0 ml of 0.9% agarose-medium and added on top of the separation layer. Small discrete colonies were easily counted after 14 days. Karyotype analysis

Fibroblasts in stationary phase were subcultured and then harvested while in logarithmic growth. When the plates contained a large number of metaphase cells, Colcemid (Grand Island Biological Co., Grand Island, New York) to a concentration of 0.4 pg/ml was added to each plate. The cells were incubated a t 37°C for 3 hours, a t which time the medium was removed and the monolayers were washed with a small volume of PBS a t 37”C, and trypsinized; the cells and PBS washings were then centrifuged together for 10 minutes a t 1,000 rpm. The supernatant was decanted and the cells slowly resuspended in hypotonic KC1 Measurement of doubling time and solution (fresh 0.075 M KCl), then incubated plating efficiency a t room temperature for 30 minutes. The cells were spun out of hypotonic soluFibroblasts from confluent cultures were seeded a t lo5 cells per 100-mm dish (day 0). tion (10 minutes as above), resuspended in Duplicate samples were trypsinized and fixative (fresh methanol/glacial acetic acid counted on a cell counter (Cellscope, Particle 3:11), and incubated a t 4°C for 30 minutes. Data Corp.) a t 24-hour intervals for 10-14 Following this treatment, they were spun days. Cultures were fed every second day. One again (as above), resuspended in 0.5 ml of fixacontrol and one patient strain were always tive, dropped onto cold slides (about 4°C) and tested concurrently using the same medium, air-dried. The slides were stained for 4.5 and serum and incubator. Doubling times minutes with freshly prepared Giemsa stain were determined from the exponential portion (0.15 N NH,OH: Giemsa: distilled water: 3:7:60). Preparation was completed by 10 dips of such graphs. To measure plating efficiency confluent fi- in each of two containers of acetone, 10 dips in broblasts were seeded in appropriate numbers acetone: xylene: 50:50, 10 dips in each of 2 (100-400 cells per 100-mm dish) and incubated containers of xylene and finally mounting for 2 weeks (controls) or 3 weeks (patients). with Permount. In some cases, to improve the Because of the time difference, the colonies in quality of the stained preparation, acid hydrothe FA strains were larger and could be iden- lysis (1 N HC1, 6OoC) was carried out for 5 tified more readily, but the numbers of col- minutes just prior to Giemsa staining. onies were not different in 2- and 3-week inAccumulation of metaphases cubations of either type of cell strain. Plates Confluent fibroblasts were subcultured and were stained by pouring off the medium and adding 1%methylene blue (Fisher Scientific when in logarithmic growth (40 hours later), Co.) in methanol: water (1:l) for 30 minutes they were treated with 0.4 pg/ml Colcemid. followed by several rinses with tap water. Cells were harvested a t various times from 0Plates were scanned with a dissecting micro- 60 hours after the addition of Colcemid. The scope and colonies scored when they contained method used for harvesting the cells was the same as that used for karyotype analysis. 32 cells or more. For lymphoblast plating a confluent fibro- Cells in metaphase as well as cells containing blast “feeder” layer in a 60-mm dish was micronuclei were scored as “metaphases.” overlaid with 2.5 ml of complete a-medium Data obtained in this way can be used to estimate the generation time of a culture by containing 0.9% of low gelling temperature

314

WEKSBERG, BUCHWALD, SARGENT, THOMPSON AND SIMINOVITCH TABLE 2

Comparison of division probability (Pi estimates for control and F A strains Control

FA

t statistic

Type of experiment Mean i S.D. (N) Mean

Plating efficiency (%) Estimated P Maximum accumulated metaphases (%) To (hr) Estimated P Doubling time (TD hr) TDlTo Estimated P

2

S.D. (N) Value

d.f.

D

45.6t4.6(8) 0.77-tO.017

16.4'4.2(11) 0.63'0.026

13.5

17

< 0.001

5554(3)

2 2 2 lO(3)

5.3

4

< 0.01

10.7

12

< 0.001

16.2t 0 . 3 0.78t0.020

24.551.8(6) 1.51t0.11 0.80-tO.03

22 f1.7 0.61 '0.046 45.524.5(8) 2.06t0.22 0.6920.02

N refers to the number of strains examined. The mean value of P from these three measurements is 0.78 2 0.015 for controls and 0.64

* 0.042 for FA. This difference is significant (p < 0.01, d.f. = 4).

growth phase, fresh medium containing various concentrations of drugs was added to a series of dishes and the cultures were incuDetermination of generation time by bated for 24 hours. The cells were then washed 3H-TdR labelling with PBS, trypsinized, counted by cell counter Confluent fibroblasts were subcultured into and plated in appropriate numbers to test for a-medium with 15%fetal calf serum, contain- survival as described above. The exponential ing no supplements of deoxy- or ribonucleo- portion of survival curves was drawn by eye, sides. When the cells were in logarithmic and a representative random sample proven growth phase, they received a 1-hour pulsc by regression analysis. The agents used were EMS and MNNG (Sigof thymidine- [ m e t h ~ l - ~ H (3H-TdR, I 2.5 pCi/ ml, 210 Ci/mmole; New England Nuclear, ma Chemical, St. Louis, Missouri), MMC Boston). When the radioactive medium was (Schwarz/Mann., Orangeburg, New York), arremoved, cultures were washed twice with tinomycin D (General Biochemicals, Chagrir PBS and medium supplemented with nucleo- Falls, Ohio), and caffeine (Eastman Kodak, sides (10mg/l each of 4 deoxyribosides and 4 Rochester, New York). ribosides) was added. Cells were harvested a t Measurement of survival of lymphoblasts various intervals following the pulse and preon exposure to MMC pared as for karyotype analysis, but not stained. Exponentially growing lymphoblasts from Autoradiography was performed by coating roller tubes were centrifuged and the medium slides with NTB-2 Nuclear Track Emulsion removed and replaced with PBS containing (Eastman Kodak, Rochester, New York) di- the appropriate concentration of MMC. The luted with distilled water (2:3). The slides cells were incubated in the dark for 1hour and were allowed to dry for several hours, then MMC was quickly removed by washing twice packed in plastic light-tight boxes and stored with PBS. The cells were then plated to deterat 4°C for 7 days. The slides were developed for mine survival as described above. 2 minutes in D-19 developer (Kodak, Toronto), Measurement of survival to treatment with rinsed in water and treated for 2 minutes with psoralen plus long wave length UV Rapid Fixer (Eastman Kodak, Rochester, New York). The slides were then air-dried and Fibroblasts were seeded a t 2-4 x lo5 cells/ stained with Giemsa as for karyotyping. Back- 100 mm dish and grown to the logarithmic ground grain counts were assessed with blank phase. Prior to irradiation, the culture meemulsion-coated slides. M dium was removed and PBS containing 8-metoxypsoralen (Sigma) was added. The Measurement of survival of fibroblasts plates were incubated in the dark for 30 to exposure to drugs minutes a t OOC. Cells were then exoosed a t 0°C Confluent cultures were subcultured using to a Blak-ray long wave ultrabiolet lamp 1:8 dilutions. When cells were in logarithmic model UVL-56 (Ultra-Violet Products, Inc.).

measuring the time required to obtain a maximum yield of metaphases.

315

CELLULAR DEFECTS IN FANCONI ANEMIA

100 0

.

M v)

rn

r a rn

r” Y

10

&?

I

I

I

I

I

t

10

20

30

40

50

60

Hours after colcemid addition Fig. 1 Metaphase accumulation curves of one control and one FA strain.

The incident intensity a t the sample surface was 70 pW/cm2as measured by a Blak-ray ultra-violet meter model 5-22 (Ultra-Violet Products, Inc.). After irradiation for the appropriate length of time cells were washed twice with PBS, and then trypsinized and plated for determination of survival. Rapidly growing cultures of lymphoblast cells in roller tubes were used for similar survival studies. The cells were centrifuged and t h e medium replaced with PBS containing M 8-methoxypsoralen. Aliquots of the suspension (0.5-1 x lo6 cells/ml) were distributed into 60-mm dishes which were then held a t 0°C for 30 minutes. The irradiation procedure was the same a s for fibroblasts. The treated cells were transferred into tubes, centrifuged and washed twice with PBS. They were then plated in t h e same manner used to investigate survival to MMC treatment. RESULTS

Growth properties o f fibroblasts from FA patients As reported by Elmore and Swift (’76) FA fibroblast strains have a mean population doubling time of 30 hours while controls have a value of 23. The prolonged doubling time could

mean t h a t (a) a certain fraction of FA cells is “dying” at each division, i.e., is unable to proceed through another division; or that (b) the generation time of individual cells from patients is longer than t h a t of cells from controls, or both. To determine which of these problems is present in FA cells, we have compared the growth rate, plating efficiencies and other growth characteristics of FA and normal cells and have assessed these measurements in terms of a theoretical model of population dynamics developed by Pujara (‘64). In this model, the growth data obtained should all be related to a value (P) which expresses the probability that a cell formed in a given mitosis will complete the next division successfully. Comparison of plating efficiencies of cells FA patients and controls

We first compared the plating efficiencies (PE) of FA and normal cells, using cells between generations 8 and 14. As may be seen in table 2, the PE of FA cells was significantly lower than that of controls (16.4%vs. 45.6%; t = 13.5, p < 0.001). Using Pujara’s (’64) theoretical relationship between PE and division

316

WEKSBERG, BUCHWALD, SARGENT, THOMPSON AND SIMINOVITCZI

probability P, the P values associated with the PE's were found to be 0.77 and 0.63 for normal and FA cells respectively (table 21, showing that FA cells have a reduced probability of proceeding to t h e next division as compared to controls. Comparison of cell division potential of FA and normal cells by measurement of metaphase accumulation If a culture contains a significant number of dying (nondividing) cells this situation is reflected in t h e decreased number of metaphases accumulating in such cultures in the presence of a mitotic inhibitor such as Colcemid. Therefore, the cell division capacities of FA and normal cells were next compared by growing the cultures for a t least one doubling time in medium containing Colcemid and measuring the fraction of accumulated metaphases. In order to provide a positive control, comparative studies were also carried out with a Chinese hamster-ovary (CHO) cell line, in which significant cell death would not be expected. The maximum in accumulated metaphases for CHO cells was reached about ten hours after addition of Colcemid, and was found to be 85%.From Pujara's ('64) theoretical treatment, the fraction of cells undergoing one division is given by 2P-1 and therefore P in this case equalled 0.93. The metaphase accumulation curves for one FA strain and one control are shown in figure 1. The maximum accumulation reached by control human diploid fibroblasts in the first 45 hours following the addition of Colcemid was about 55%. With the FA strain, sampling was continued for 70 hours after addition of Colcemid and a maximum of only 20%was obtained. As shown in table 2, the average maximum attained by control human diploid fibroblasts and FA cells was 55.3% and 22.0% respectively. The P values calculated from these data by the method of Pujara ('64) were 0.78 (range 0.76-0.80) for normal and 0.61 (range 0.57-0.66) for FA cells, respectively. The results are statistically significant (p < 0.01) for maximum accumulated metaphase, P value and To (see below). These P values are similar to those estimated from the PE data, and again show t h a t FA cells have a decreased probability of further division compared to normal human fibroblasts. Comparison of generation times of cells from normal and FA strains The measurements of both PE and meta-

phase accumulation indicated that FA cells have a defect which reduces their reproductive ability (P value). It was next of interest to determine whether FA cells also show a longer generation time than normal cells. Generation times (To) can be obtained in two ways: (a) by counting metaphase cells accumulated at specific times after addition of mitotic poisons, or (b) by counting radio-labelled metaphases over time after a short pulse with 3H-TdR. Using the first method, a value for To was derived from data such as that shown in figure 1by estimating the time required to accumulate the whole population in metaphase. This average value was 16.2 hours (range 16-17) for control cells and 22.0 hours (range 21-24) for FA cells (table 2). The results obtained after a 3H-TdR pulse for one FA strain and one control are shown in figure 2. From these data we have calculated the duration of each portion of the cell cycle for normal and FA cells. For the control strain, Towas 16.5 hours consisting of G = 5.5, S = 6 and Gz M = 5 hours while for the FA cells G, = 5, S = 8, and G 2 M = 8 hours for a To = 21 hours. Although only one FA and one normal strain have been examined by this latter method, the values obtained agree well with the results of the metaphase accumulation experiments, and support the contention that Tois extended in FA strains.

+

+

Estimation of TdT,for normal and FA cells The data provide two estimates of the value of P for FA and normal cells. Pujara ('64) showed that a third estimate of the P value could be obtained by measuring the ratio of population doubling time to generation time (TD/TJ. In order to determine P using this method, i t was necessary to measure doubling times (TD) for both normal and FA cells (see MATERIALS AND METHODS and the data shown in table 2). The average doubling time for normal cells was 24.5 hours whereas for FA cells it was 45.5 hours (t = 10.7, d.f. = 12, p < 0.001). In calculating TD/To we assumed t h a t the generation times for all the strains would be similar and used a single value (16.2 hours for control, 22 hours for FA strains) in the subsequent calculations. The values of P were then obtained using Pujara's ('64) curve relating TD/T, to P. Table 2 also shows that P values for FA cells estimated in this way are again less than those for controls and in both cases the values are similar to those obtained by the other two methods.

317

CELLULAR DEFECTS IN FANCONI ANEMIA

66

o FA-151 * C -57

60 m W m

54

m c a m Y

48

E

42

73 W W

-

36

n

m -

30

+

24

C

a,

z W

Q

18 12

'.

6 "

3

6

12

18

24

30

36

Hours after 3H-TdR pulse Fig. 2 Labelled metaphaws in cnnlrol xnd FA strains a s a function of time after the addition of a 3H TdR pulse.

Thus the P values obtained from the three different types of experiments (table 2) show t h a t the division probability of FA cells is considerably reduced as compared with normal cells, no matter which method is used for measurement.

Sensitivity to mutagens o f fibroblasts from FA patients and controls To determine whether differences in sensitivity to alkylating agents could be demonstrated between FA and normal cells, we began by examining the response of FA cells to MMC, a bifunctional alkylating agent which induces DNA crosslinks (Iyer and Szybalski, '63). Fibroblasts cultures were treated with MMC and colony survival scored as described in MATERIALS AND METHODS. For all strains t h e resulting curves were simple exponentials but the slopes for the FA cells were all less than those of controls (fig. 3A). The curves in figure 3A were characterized by a parameter Dlo, t h e dose required to reduce survival to 10% (Elkind and Whitmore, '67). The mean D,, of nine controls was 0.022 pg/ml (with an S.E.M. of 0.002) and values ranged between 0.016 and 0.029. The D,, values for

each patient were then compared to the mean of controls to yield an index of patient sensitivity @.I. = mean of control Dlo/patient Dlo)as shown in table 3. Although FA strains showed variation in sensitivity to MMC all were more sensitive than controls and S.I. ranged from 3 to 27. One patient with aplastic anemia had a MMC sensitivity similar to that for controls (D,, = 0.02 pg/ml). Although there were major differences in sensitivities between patients, repeated measurements on one strain gave reproducible results. In addition, there are two instances in which a patient donated a second cell strain two years after the first and in both cases the S.I. of the second strain was similar to the first (table 3, patient RS and RG). These findings indicate that MMC sensitivity is a stable genetic property of the patient, unlike the rate of chromosome breaks which fluctuates over time (Berger et al., '75). Further support for the significance of MMC sensitivity in FA comes from the finding that the D I oof strain FA 145 was similar to that of the other FA patients. This strain came from a 2-year-old boy who has all the early indications of FA, but has not yet developed pancytopenia. We

318

WEKSBERG, BUCHWALD, SARGENT, THOMPSON AND SIMINOVITCH

B

10-3

tI

*nsc 3 .HSC 4 9 O H S C 72 @HSC 9 9 I

I

I

I

,001

.005

.01

.013

10-3

1

I

I

I

I

SO

1 .o

1.5

2.0

MlTOMYClN C CONCENTRATION u g / m l

Fig. 3 Survival of normal and FA fibroblasts (A) and lymphoblasts (B)after exposure to Mitomycin C. In t h e absence of the drug, the plating efficiency of fibroblasts was in the range described i n table 2 and that of lymphoblasts i n t h e range 5.20%for both FA and controls.

plan to follow this child to see whether the level of MMC sensitivity remains stable with the onset of pancytopenia. Sensitivity of lymphoblast lines to mutagens

The biochemical analysis of the FA phenotype would be facilitated considerably by the availability of transformed lymphoblast cultures since such lines do not show senescence; it was thus of interest to determine whether lymphoblast lines also showed increased sensitivity to MMC. As may be seen in figure 3B where cells from two FA and two control individuals are compared, the lymphoblasts from the FA patients again show an increased MMC sensitivity. Furthermore, the extent of increase in sensitivity of the lymphoblasts from patients MB and SW paralleled the increase observed when fibroblasts from these patients were examined (table 3).

sensitivity to psoralen and long wave length UV

MMC is known to cause DNA crosslinks and the greater sensitivity of FA cells to this drug may be due to an inability of FA cells to repair such crosslinks. If this were the case, then treatment of FA cells with other agents known to induce crosslinks should result in greater sensitivity than control strains. We therefore tested both fibroblasts and lymphoblasts for their sensitivity to exposure to psoralen and long wave length UV, a treatment also known to produce DNA crosslinks (Cole, '70). Two FA strains representing patients with high and intermediate sensitivity to MMC were studied along with two controls. In the absence of UV irradiation neither the FA or control cells were killed by psoralen (data not shown). Combined treatment with psoralen and radiation yielded the survival

3 19

CELLULAR DEFECTS I N FANCONI ANEMIA 1

10

S/SO

10

.

HSC 49 HSC 3 HSC 62 o HSC 72 A

10

2

6

4

2

4

6

M i n u t e s of e x p o s u r e

Fig. 4 Survival of normal and FA fibroblasts (A) and lymphoblasts (B) after treatment with psoralen and long wave length UV. Fibroblast and lymphoblast plating efficiencies were similar to those in table 2 and figure 3.

TABLE 3

Sensitiuity of Fanconi anemia cells to crosslinking agents PS + uv

MMC Patient

EP LL RS MC

sw CB MB RG

Fibroblast strain

D,o (pg/ml)

FA-145 FA-61 FA-52 FA-182 FA-400 FA-402 FA-421 FA-156 FA-61 FA-151

0.0010 0.0060

0.0045 0.0050 0.0040 0.0030 0.0020 0.0010 0.0009 0.0008

D,a S.I.

3.1 3.1 4.9 4.4

5.5 1.3 11 22 24.4 21.5

(mid

S.I.

MMC Lymphoblast strain

D,O (pg/ml)

PS+U V

S.I.

D,, (mid

S.1

HSC62

-

HSC99

0.54

-

-

HSC72

0.089

-

-

T h e D,ofor each strain was determined from graphs such as those illustrated in figures 3 and 4. S.I. (sensitivity index) is t h e ratio of the mean of the D,, of control strains to t h e D,oof the appropriate strain. T h e mean D Iofor controls was 0.022 pglml (MMC-fibroblasts), 1.25 pglml (MMC-lymphoblasts), and 4.6 minutes (PS-UV fibroblasts and lymphoblasts).

320

WEKSBERG, BUCHWALD, SARGENT, THOMPSON AND SIMINOVITCH

curves shown in figure 4. As can be seen, the FA fibroblasts and lymphoblasts show an increased sensitivity to the treatment. A significant shoulder was observed in the curve for the control strains. In FA strains, on the other hand, only a small or non-existent shoulder was seen. The slope of the exponential portions of the control and FA curves were similar, however, suggesting that FA cells lacked the capacity for sublethal repair of the damage caused by psoralen and UV. FA strains with greater sensitivity to MMC (e.g., FA 421) also showed higher sensitivity to psoralen and UV while those with intermediate sensitivity to MMC (e.g., FA 400) manifested a reduced sensitivity to psoralen and UV (table 3). Similar correlations were also seen with lymphoblast lines (table 3). These results support the hypothesis that the degree of sensitivity to DNA cross-linking agents is a stable property of FA cells, characteristic of the donor.

Sensitivity to other agents We examined at least one patient considered very sensitive to MMC and one of intermediate sensitivity for sensitivity to several concentrations of other drugs. FA cells did not respond differently from controls to EMS (up to 300 pg/ml, 24 hours exposure) a monofunctional alkylating agent which does not produce significant numbers of crosslinks (results not shown). Similarly, no difference in response to MNNG (to 5 pg/ml) or to actinomycin D (to 0.07 pg/ml) could be found between FA and control cells (results not shown). DISCUSSION

Our study of the growth parameters of FA cells in culture has shown that these cells have an increased generation time and an increased rate of cell death. Both these factors must contribute to the increased doubling time observed in strains derived from FA patients. The increased probability of cell death in cells from FA patients may explain their stature and low birth weight and the type and number of congenital malformations seen in such patients are probably randomly determined by failure to support rapid growth. Therefore, the heterogeneity seen in FA patients in terms of age of onset, type and number of congenital malformations, stature, and the degree of the hemopoietic problems may be understood in terms of a stochastic model of cell death superimposed upon possible heterogeneity in gene expression.

The increase in generation time was found to be due to prolonged S and G, periods. Although only one cell strain was tested, this finding is supported by the results of two other studies. Sasaki ('75) has shown t h a t lymphocytes derived from one patient with FA had increased S and G 2 periods compared to control lymphocytes and Loughman ('73) has found a n unusually large proportion of cells in G2, in short-term lymphocyte cultures from FA patients. Since the spontaneous chromatid breaks seen in FA cells must occur late in S or in GZ,the breakage may be related to the prolongation of S and G, in FA cells. The increased S and Gztimes may also be involved in the increased SV40 transformation seen in vitro (Todaro et al., '66) and the increased malignancy in vivo. An extended Gzperiod has been found in fibroblast cultures derived from patients with Down syndrome (Paton et al., '74). Such patients also have an increased risk of malignancy in vivo (German e t al., '62) and an increased rate of transformation by SV40 in vitro (Todaro and Martin, '67). The experiments described in this paper and others show that FA cells exhibit increased sensitivity to DNA crosslinking agents (MMC and psoralen plus long wave length UV). This finding is not limited to a specific cell type, since the data from fibroblasts and lymphoblasts are similar. Sensitivity to MMC differed between patients, suggesting that there is heterogeneity within the disease, as for Xeroderma Pigmentosum (XP) (Cleaver, '72). It will be interesting to determine whether, as in XP, this reflects discrete levels of repair corresponding to different complementation groups. The sensitivity of FA cells to MMC and psoralen plus long wave length UV is highly specific, since FA cells do not show any increased sensitivity to EMS, actinomycin D or MNNG. The fact that EMS does not show any differential activity towards FA as shown in our study and that of Fujiwara et al. ('77), iridicates that the increased sensitivity is specific for bifunctional rather than monofunctional alkylating agents. This conclusion is supported by the observations of Sasaki and Tonomura ('73) that there was no increase in chromosome breakage when FA cells were treated with decarbamyl mitomycin C , the monofunctional derivative of MMC, and of Auerbach and Wolman, ('76) who found t h a t nonlethal doses of the bifunctional alkylating agent diepoxybutane increased chromosome breaks in FA cells but not in controls.

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The primary defect in FA cells may involve either DNA replication or repair (German, '72) and the findings on growth suggest a temporary block or slowdown in DNA replication. Capacity for in vitro growth is undoubtedly determined by a large number of genes. Decreased growth has also been reported in fibroblast strains from patients with progeria (Danes, '711, Werner syndrome (Martin et al., '701, Cockayne syndrome (Hoar, personal communication) and Bloom syndrome (unpublished results). These diseases are characterized by a number of biochemical abnormalities related to reduction of growth potential. In Werner syndrome, cultured cells have been shown to have a retarded rate of DNA replication although several tests of DNA repair following UV and y-radiation were normal. In Bloom syndrome, Hand and German ('75) reported a 26% reduction in the rate of DNA chain growth. Cells from these patients have been reported to show increased UV sensitivity (Giannelli et al., '77) and possibly mitomycin C sensitivity (unpublished results). Cells from patients with Cockayne syndrome have also been shown to have defective excision repair (Hoar and Waghorne, '78). The results on damage with alkylating agents provide strong evidence that FA cells are deficient in the repair of DNA crosslinks. Latt et al. ('75) have provided further support for this conclusion. Following MMC treatment, FA cells do not exhibit the characteristic level of sister chromatid exchange (SCE) seen in normal cells. Since recombination is know to be an absolute requirement for the repair of crosslinks in E. coli, the data of Latt et al. ('75) suggest that FA cells have difficulty in carrying out the recombinant aspect of repair following MMC treatment. There is some contrary evidence, however. Although no increase in chromosome breaks following EMS treatment could be demonstrated by Sasaki ('751, others have shown t h a t EMS induces a greater number of breaks in FA cells than in controls when exposure is extended to six days (Auerbach and Wolman, '76). In bacterial models, susceptibility to damage by one agent may be associated with susceptibility to another, suggesting a common step in the repair process. For example, E. coli uvr or rec mutants sensitive to MMC are also sensitive to UV light, indicating that both gene products normally are required to deal with either type of damage (Grossman et al., '75). When similar studies have been con-

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ducted with FA cells, the results are inconsistent. In one report, pyrimidine dimer excision was normal, and the cells were UV-resistant (Regan et al., '70). However, Poon et al. ('74) found that a t doses greater than 150 ergs mm-2, FA cells strain were found to exhibit a deficiency in UV-induced repair. In these experiments, unscheduled DNA synthesis was normal and the incision step was normal, but the dimers were not excised. The authors interpreted this to mean that FA cells have an exonuclease deficiency. However, they could not detect this deficiency below the high doses of 150 ergs mm-2. The results of studies on y-radiation of FA cells are equally inconsistent. Remsen and Cerutti ('76) investigated the repair of base damage following y-radiation. They found that two out of four patients with FA exhibited a decreased rate of excision of the reaction product from the DNA substrate. We have found (unpublished results) that cells from FA patients have a decreased capacity to repair sublethal damage on treatment with yradiation. However, no consistent increase in sensitivity could be demonstrated, an observation also reported by Sasaki ('78). In addition, Sasaki and Tonomura ('73) were unable to confirm an increase in chromosome breakage in FA lymphocytes following y-radiation, previously reported by Higurashi and Conen ('73). In summary, there is considerable evidence that FA cells have a defect in the repair of DNA crosslinks. The defects in the handling of y-ray and UV damage, if they are operative in vivo, may be due either to one enzyme taking part in two different repair systems or to similar damage caused by different agents, and, therefore, recruitment of the same enzyme. Both Clever ('77) and Sasaki ('78) have proposed a model for the repair of DNA crosslinks or closely spaced damaged sites induced by high doses which could explain the decreased number of induced sister chromatid exchanges (Latt et al., '751, increased number of chromosome aberrations and decreased frequency of mutants (R. Weksberg, M. Buchwald, P. Sargent and L. Siminovitch, unpublished results) seen in FA cells. There is some evidence that cells from patients with other diseases, such as XP and Ataxia telangiectasia also show increased sensitivity to crosslinking agents (Baden et al., '72; Taylor et al., '75; Hoar and Sargent, '76). Such findings imply that the repair of crosslinks is an exceedingly complex process requiring a variety of different enzyme sys-

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tems. This is in keeping with studies on bacteria, in which the repair of crosslinks requires both the uvr and rec enzyme systems (Cole, ’71). Hopefully, the study of human chromosomal breakage syndromes will help to elucidate common DNA repair pathways in mammalian cells, and may give insight into the role of defective DNA repair in malignant transformation. ACKNOWLEDGMENTS

We thank Sophie Asvestos for technical assistance, Dr. Gary Jones for many helpful discussions, and Dr. P. Quinn for the mycoplasma tests. R. W. was supported by studentships from the Medical Research Council of Canada (MRC) and the Muscular Dystrophy Association of Canada. The research was supported by grants from MRC and NIH-NCI (USA). LITERATURE CITED Auerbach, A. D., and S. R. Wolman 1976 Susceptibility of Fanconi’s anemia fibroblasts to chromosome damage by carcinogens. Nature, 261: 494-496. Baden, H. P., J. M. Parrington, J. D. A. Delhanty and M. A. Pathak 1972 DNA synthesis in Normal and Xeroderma Pigmentosum Fibroblasts following treatment with 8methoxypsoralen and long wave length ultraviolet light. Bioch. Bioph. Acta., 262: 247-255. Bachetti, S., and G. F. Whitmore 1969 Actinomycin D: Effects on mouse L-cells. Biophys. J., 9: 1427-1445. Berger, R., A. Bussel and C. Schenmetzler 1975 Anomalies Chromosomiques et Anemie de Fonconi. Etude de 4 Cas. Nouv. Rev. Fran. d’Henatol, 15: 539-550. Cleaver, J. E. 1969 Xeroderma Pigmentosum: A human disease in which an initial stage of DNA repair is defective. Proc. Natl. Acad. Sci. (U.S.A.), 63: 428-435. 1972 Xeroderma Pigmentosum: Variants with normal DNA repair and normal sensitivity of Ultraviolet light. J. Invest. Dermat., 58: 124-128. 1977 DNA Repair process and their impairment in some human diseases in progress in genetic toxicology. In: Progress in Genetic Toxicology. D. Scott, B. A. Bridges and F. H. Sobels, eds. Elsevier/North-Holland, Amsterdam, pp. 29-42. Cole, R. S. 1970 Light induced cross-linking of DNA in the presence of furocoumarin psoralen. Studies with phage lambda, Escherichia coli and mouse leukemia cells. Bioch. Biophys. Acta., 21 7: 30-,39. 1971 Inactivation of Escherichia coli F episomes a t transfer and bacteriophage lambda by psoralen plus 360 mm light: significance of deoxyribonucleic acid crosslinks. J. Bacteriol., 107: 846-852. Danes, B. S. 1971 Progeria: a cell culture study on aging. J. Clin. Inv., 50: 2000-2003. Elkind, M. M., and G. F. Whitmore 1967 The Radiobiology of Cultured Mammalian Cells. Gordon and Breach, New York. Elmore, E., and M. Swift 1976 Growth of cultured cells from patients with Fanconi anemia. J. Cell. Physiol., 87: 229-239. Fanconi, G. 1967 .Familial constitutional panmyelogytopathy, Fanconi‘s anemia (FA). I. Clinical aspects. Seminars Heqat., 4: 233-240.

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Specific cellular defects in patients with Fanconi anemia.

Specific Cellular Defects in Patients with Fanconi Anemia ROSANNA WEKSBERG, MANUEL BUCHWALD, ' PATRICIA SARGENT, MARGARET W. THOMPSON AND LOUIS SIMINO...
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