Arch Dermatol Res (1991) 283:449-455
Archives of
9 Springer-Verlag 1991
Defects in antioxidant defense and calcium transport in the epidermis of xeroderma pigmentosum patients K. U. Schallreuter 1, M. R. Pittelkow 2, and J. M. Wood s 1 Department of Dermatology, University of Hamburg, Martinistrasse 52, W-2000 Hamburg 20, Federal Republic of Germany 2 Department of Dermatology, Mayo Clinic, Rochester, Minn., USA 3 Department of Biochemistry, University of Minnesota, St. Paul, Minn., USA Received April 13, 1991
Summary. A comparative study of the antioxidant enzymes superoxide dismutase, catalase, glutathione reductase and thioredoxin reductase was undertaken in two families with xeroderma pigmentosum (XP) and in healthy controls of corresponding skin phototypes. Epidermal blister roofs obtained from the XP patients revealed significant decreases in catalase, thioredoxin reductase, and superoxide dismutase, but glutathione reductase was unaffected. In addition, keratinocytes established from XP patients contained a significantly higher than normal intracellular calcium concentration compared with control cells from a corresponding skin type. Keratinocytes established from an XP obligate heterozygote revealed intermediate levels of calcium between XP homozygotes and controls. Previously high intracellular calcium has been shown to compromise the redox status of keratinocytes by allosteric inhibition of the thioredoxin reductase]thioredoxin electron transfer system. In XP homozygous keratinocytes from sun-exposed epidermis, the intracellular concentration of reduced thioredoxin was decreased to 50% compared with these cells from unexposed skin. Taken together, the results from this study indicate that the epidermis in XP patients lacks effective defense against free radicals and peroxides. In addition to the well-established defect in the normal rates of unscheduled DNA repair, these findings provide an even better explanation for the multiple cutaneous neoplasms in these patients. Key words: Antioxidant enzymes - Calcium transport Xeroderma pigmentosum
Xeroderma pigmentosum (XP) was first described by Hebra and Kaposi in 1874 [14]. The disorder represents a rare and heterogeneous group of diseases with an increased sensitivity to sunlight followed by a high frequency of sun-induced malignancies in the exposed skin Offprint requests to: K. U. Schallreuter
areas [14]. The typical clinical symptoms are a poikilodermatic skin with dyschromia, atrophy and telangiectasias with constant progression and development of multiple benign and malignant neoplasms. Most of these abnormalities arise from keratinocytes as precancerous lesions, as basal cell carcinoma and squamous cell carcinoma, whereas lentigo maligna arises from melanocytes and is the precursor for lentigo maligna melanoma. The overall frequency of this autosomal recessive disorder is approximately 1 in 250000, with a higher expression ( 1 0 - 2 5 in 100000) in Japan, Holland and Israel [14]. Two forms of XP have been discovered. In one form, a lack of those enzymes responsible for normal rates of unscheduled D N A synthesis occurs, and in the variant form, unscheduled DNA repair is normal, but the postreplicative maintenance of D N A integrity is absent [25]. In the excision-deficient group, there are nine complementation types ( A - I ) and each type is believed to have a distinct defect in D N A repair [2]. The majority of XP patients have the variant form of the disease and the complementation groups A, C, D, G and I are extremely rare [6, 7, 16]. Recently, it has been demonstrated that X-irradiated fibroblasts from XP heterozygotes yielded a two-fold increase in the frequency of chromatid breaks and gaps compared with control cells [16]. It has also been shown that XP cells are deficient in antioxidant defense against electrophiles generated by fluorescent light [24, 26], alkylating agents [13, 23] and, in some cases, ionizing radiation [4]. Very early studies of the epidermis of XP patients by electron microscopy showed significant membrane degeneration especially in mitocho~dria, Golgi apparatuses and other intracellular organelles. The exceptions were the melanosomes which appeared to be very resistant to UV-photochemical oxidative stress [22]. A study of 31 families of XP patients revealr that obligate heterozygotes in XP also have an increased predisposition to non-melanoma skin cancers compared with the normal population [35]. Therefore, even XP heterozygotes appear to be more susceptible to UV-mediated D N A damage.
450
K. U. Schallreuter et al.: Antioxidant defense and calcium transport in epidermis in xeroderma pigmentosum
The majority of molecular biology, genetic and biochemical studies on XP have been conducted with cultures of dermal fibroblasts. To date, there are only a few reports on keratinocytes and melanocytes which actually undergo oxidative stress and malignant conversions. Recently, the metabolic pathways for UV-generated oxygen radicals in the h u m a n epidermis have been reviewed [30]. Key enzymes for superoxide anion radical (O2) reduction have been identified as superoxide dismutase (SOD) and the thioredoxin reductase/thioredoxin (TR/T) electron transfer system [30]. SOD levels are low in the h u m a n epidermis c o m p a r e d with h u m a n liver and kidney [3]. This could be due to the conversion of 0 2 by SOD to hydrogen peroxide ( 0 2 - ) . This product is extremely toxic to cells in the epidermis through its UVphotochemical reaction yielding reactive hydroxyl radicals (OH-) [30]. OH. radicals are k n o w n to oxidize proteins, lipids and nucleic acids in a r a n d o m fashion [33]. The T R / T system in h u m a n keratinocytes represents 5% of the total acidic protein in the cell cytosol fraction [32]. The T R / T system (I) reduces O2 via 022- to water, and (II) regulates melanin biosynthesis [37]. This system is under allosteric regulation by calcium which binds to a single EF-hands site on T R to inhibit the electron transfer to oxidized thioredoxin (ToO [29, 32]. Therefore, the calcium status of the epidermis influences its oxidation status with high intracellular calcium leading to a decrease in antioxidant defense [32]. Two other enzymes play a m a j o r role as catalytic antioxidants in the h u m a n epidermis by metabolizing O22-. Catalase (CAT) converts 0 2- to molecular dioxygen, and the glutathione reductase/glutathione/glutathione peroxidase ( G R / G / G P ) system reduces O 2 - to water [30]. U n d e r normal conditions, the above four enzyme systems would provide adequate defense against O2 and 022-. However, an excess of UV-generated O2 deactivates catalase by irreversible oxidation of the four tetrapyrrole rings on the heme prosthetic group [1]. It is well established that some epidermis f r o m patients with XP contains higher than normal concentrations of hydrogen peroxide [36]. Keratinocyte cell cultures f r o m three XP patients revealed low catalase activities compared with healthy adult controls. Catalase was depressed even further in these ceils u p o n exposure to U V radiation. In addition, an increase in malignant conversion occurred [21]. Recently, it has been shown that T R is also deactivated approximately 50% by U V exposures below the minimal erythema dose ( M E D ) [34], whereas G R is mostly unaffected by U V A radiation. In experiments UVA-irradiated nude mouse skin, G R retained 85% of its control activities [12]. The aim of this study was to examine the redox status via CAT, SOD, T R and G R directly in the epidermis in h o m o z y g o u s and heterozygous XP patients.
England Nuclear, DuPont, Boston, Mass., USA. CAT, SOD and GR from human erythrocytes were purchased from Sigma Chemical Company, St. Louis, Mo., USA. TR and T were purified from human melanoma tissue by a modified method of Luthman and Holmgren [17, 28]. TR and T were resolved by FPLC on a mono Q HR 5/5 column in 0.05 M Tris-HC1 buffer, pH 7.5, using a 0-0.5 M NaC1 gradient. TR eluted at 0.12-0.13 M NaC1, calcium-bound TR (TRca2+) eluted at 0.10-0.105 M NaC1, and T at 0.3 M NaC1 (Fig. 1). 14CH3Hg incorporation into keratinocytes reduced thioredoxin (Tred) was measured according to the method of Schallreuter et al. [32]. Labelled calcium uptake experiments were performed on keratinocyte cell cultures grown in MCDB medium containing 0.1 x 10 -3 M calcium using the method of Schallreuter and Pittelkow [27]. Epidermal suction blisters were obtained from the XP family members and controls by a modified method of Pittelkow et al. [20]. Cell-free extracts were prepared from epidermal blister roofs by ultrasonication in 0.05 M Tris-HC1 buffer, pH 7.5, with a microprobe at the maximum setting (Heat Systems, Model 350 Ultrasonicator, New Brunswick, NJ, USA). Cell membranes were removed by centrifugation at 5000 rpm for 30 min at 0~ Protein concentrations were determined by the method of Kalb and Bernlohr [15]. An aliquot of 0.5 ml of extract was used for analysis by the mono Q HR 5/5 column equilibrated with 0.05 M Tris-HC1 buffer, pH 7.5, and a 0-0.5 M NaC1 gradient. Enzyme standards were chromatographed on FPLC under the conditions described above. SOD eluted at 0.075 M NaC1 and was assayed by ferricytochrome C reduction [11], CAT eluted at 0.16- 0.17 M NaC1 and was assayed by following oxygen evolution with a Clark oxygen electrode [8]. GR eluted at 0.22 M NaC1 and was assayed by the reduction of 5,5'-dithio-bis-2-nitrobenzoate (DTNB). The same method was used to assay TR [17, 28]. Membrane-associated TR was determined by nitroxide radical reduction [28, 31].
Patients and controls Family A. (Turkish, photo-skin type IV) (Fitzpatrick classification,
[10]). Father (35-years-old), mother (30-years-old), one healthy sister (10-years-old), one XP homozygote (13-years-old) (complementation group D). Case history. Boy (13-years-old) with normal physical and mental development, since age of 1 year, development of multiple freckles on sun-exposed areas; increasing numbers of basal cell carinoma and squamous cell carcinoma; no melanomas up to now; family history negative for XP. Family B. (German, photo-skin type II [10]). Mother (32-years-old),
one healthy sister (9-years-old), one XP homozygote (16-years-old) (complementation group C). Case history. Boy (16-years-old) with normal mental and physical development; since age of 2 years, increasing number of freckles on the total integument; family history negative for XP; first premalignant lesions at age 4, followed by basal cell carcinoma, squamous cell carcinoma, and at age 12, malignant melanoma; treatment with Tigason for 2.5 years without improvement in the frequency of basal cell carcinoma, squamous cell carcinoma or premalignant lesions. Controls. Six healthy probands with photo-skin type II (n = 3) and photo-skin type IV (n = 3) were used for the protocol.
Results Materials and methods Radiolabelled 14C-methyl mercuric chloride (42 mCi/mmol) was synthesized by the method of DeSimone et al. [9] and (radiolabelled 4%alcium chloride) (15mCi/mmol) was purchased from New
Table 1 summarizes a survey of the enzyme levels separated and identified f r o m epidermal blister roofs by using F P L C for b o t h families A and B. Figure 2 presents the
K. U. Schallreuteret al. : Antioxidantdefenseand calciumtransport in epidermis in xerodermapigmentosum 0.5
0.5 M NaCI
a
0.4
0.3
0.2
o.1
o.o 5
lO
15
20
25
30
0,5
0.5 M NaCI
b 0.4
E (CI
oo
0.3
o
0.2
t-
O
0.1
0.0 0
5
10
15
20
25
30
0.5
0.5M NaCI
C 0.4 -
0.3
0.2
4.0
9 !
!
3.0 o
I
z0~ "~
0.1
1.0
0.0 '~"
0
0.0
5
10
15
20
25
30
Fraction number, ml
Fig. l. a Purificationof melanoma thioredoxinreductase by FPLC (Aaso .m) full scale 1.0 A units on a mono Q column HR 5/5 in 0.05 M Tris buffer, pH 7.5, with a 0-0.5 M NaC1 gradient, This calcium-freeenzymeeluted at 0.12 M NaC1. b Purificationof 4SCalabelled melanomathioredoxin reductase (one 45Ca/TR molecule) on a mono Q columnin 0.05 M Tris buffer,pH 7.5. Calcium-bound thioredoxin reductase eluted at 0.105 M NaCI. e FPLC chromatography of 14CH3-HgC1labelledthioredoxinon a mono Q column in 0.05 M Tris buffer, pH 7.5 (A2so,~ full scale 1.0 A units). 14CH3HgCIlabelsthis proteinwitha 1: 1 stoichiometry( 9 -9 i.e.
/ 14CHaHgC1 + T
S~ ~4CH3-Hg-S.
\ SH
\ /
HS
T
451
individual FPLC data, in 0.05 M Tris-HC1 buffer, pH 7.5, for each member in family A using a 0 - 0 . 5 M NaC1 gradient. These results show considerable decreases in CAT, SOD and TR in both XP homozygotes compared with other family members and controls. Since TRca2 + can be distinguished from TR by FPLC, the levels of TRca~+ (inhibited enzyme) and TR (fully active enzyme) can be compared. As a consequence, TRca2+ has been used as a monitor for the cytosol calcium concentration at steady state in these samples [32]. Figure 3 presents the ratio TRc,2 . / T R in each individual family member. Both XP homozygotes indicate higher than normal intracellular calcium levels. For both mothers in families A and B, and for one daughter in family A, calciumlevels were high compared with control c skin. In order to test this calcium effect in more detail, keratinocytes were established from the XP homozygotes in families A and B, as well as from the mother in family B (obligate heterozygote), and the kinleticsl for 45Ca 2+ uptake were determined. Uptake rates and equilibria were compared with keratinocytes from a normal donor of skin type II. Figure 4 shows that both XP homozygotes maintain a high intracellular concentration~ of calcium at steady state, and the mother in family B (obligate heterozygote) had an intermediate level of~4SCa2+ between the skin type II control and the two XP homozygotes. FPLC analysis of keratinocyte cell extracts from the mother in family B afforded a separation of TRca2+ from TR in a ratio 5:3 (Fig. 5). This result is compatible with the analysis of the epidermal blister roof from the same patient yielding a TRca~+/TR of 2:1 (Table 1). Analysis of membrane-associated TR activities in 3 mm punch biopsies by electron spin resonance spectroscopy revealed that both XP homozygotes had significantly lower TR activities in sun-exposed skin versus unexposed skin compared with controls of similar skin type [31]. For XP patient A TR activity in sun-exposed skin was 30 units/3 mm biopsy per 10 rain and TR unexposed skin was 13 units/3 mm biopsy per 10 rain. The control skin activity for this XP patien t (skin type IV) was 20.4 _+ 3.0 (n = 6) [31]. For XP patient B TR unexposed skin was 3.5 units/3 mm biopsy per 10 min and TR sun-exposed skin was 2.5 units/3 mm biopsy per 10 min. The control skin activity for this XP patient (skin type II) was 14.6 _+ 3.0 (n = 7) [31]. (It should be noted that for patients with lower skin types (I and II), the membrane-associated TR assay has a greater standard deviation owing to lower enzyme activities.) The influence of UV radiation on the expression of intracellular TR activity in XP was evaluated further by analyses of the intracellular concentration of T~od in keratinocytes established from sun-exposed versus unexposed skin ofXP homozygote B. Table 2 shows that TR activity is significantly depressed ( - 5 0 % ) in keratinocytes from sun-exposed skin of this XP hon:tozygote compared with his unexposed skin and with cells from his mother (obligate heterozygote) and his sister. The low concentration of Trea in keratinocytes from sun-exposed skin in XP confirms local variations with more oxidizing intracellular conditions.
452
K . U . Schallreuter et al.: Antioxidant defense and calcium transport in epidermis in xeroderma pigmentosum
Table 1. FPLC separation and identification of enzymes involved in 0 2 and 022 reduction from epidermal blisler roofs taken from families A and B with XP. Enzyme levels are expressed as gg protein/rag epidermal blister-derived celt extract Subject
SOD
CAT
GR
TRca2 +
TR
Family A (XP-complementation group D): Father Mother Daugtlzer Son (XP) Type IV skin (control) mean (n = 5)
6.53 5.25 656 1.6 6.2 _+ 0.5
12.45 12.25 14+05 8.0 12.5 _+ 1.2
3.06 5.0 n_ d. 4.8 4.7 _+ 0.4
5.1 4.0 4.2 2.4 5.60 _+ 0.2
4.7 2.5 2.16 1.6 5.1 _+ 0.75
Family B (XP-complementation group C): Mother Son (XP) Type II skin (control mean (n = 5)
4.1 2.0 3.8 + 0.35
10.5 1.4 12.1 _+ 0.8
3.6 3.9 3.7 _+ 0.4
5.0 2.6 4.6 -t- 0.3
2.5 1.5 3.70 _+ 0.80
b1
tt II
003 4!
0.03
mc,,~ CATI I
- 0,5M NaCL
0.5M NaCI 0.02-
0.02 -
I
',j m(
0.01 000 o,I U.l 0 Z
0
~i
o
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10
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I
~,
J ,
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0
3~3
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;
;o
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2b
g
30
d
I I
II II II II II
| I l I SOD
; TRc2. CAT I 0,02-
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0
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I I I
0.03-
GR
0.01
I
Archives of
9 Springer-Verlag 1991
Defects in antioxidant defense and calcium transport in the epidermis of xeroderma pigmentosum patients K. U. Schallreuter 1, M. R. Pittelkow 2, and J. M. Wood s 1 Department of Dermatology, University of Hamburg, Martinistrasse 52, W-2000 Hamburg 20, Federal Republic of Germany 2 Department of Dermatology, Mayo Clinic, Rochester, Minn., USA 3 Department of Biochemistry, University of Minnesota, St. Paul, Minn., USA Received April 13, 1991
Summary. A comparative study of the antioxidant enzymes superoxide dismutase, catalase, glutathione reductase and thioredoxin reductase was undertaken in two families with xeroderma pigmentosum (XP) and in healthy controls of corresponding skin phototypes. Epidermal blister roofs obtained from the XP patients revealed significant decreases in catalase, thioredoxin reductase, and superoxide dismutase, but glutathione reductase was unaffected. In addition, keratinocytes established from XP patients contained a significantly higher than normal intracellular calcium concentration compared with control cells from a corresponding skin type. Keratinocytes established from an XP obligate heterozygote revealed intermediate levels of calcium between XP homozygotes and controls. Previously high intracellular calcium has been shown to compromise the redox status of keratinocytes by allosteric inhibition of the thioredoxin reductase]thioredoxin electron transfer system. In XP homozygous keratinocytes from sun-exposed epidermis, the intracellular concentration of reduced thioredoxin was decreased to 50% compared with these cells from unexposed skin. Taken together, the results from this study indicate that the epidermis in XP patients lacks effective defense against free radicals and peroxides. In addition to the well-established defect in the normal rates of unscheduled DNA repair, these findings provide an even better explanation for the multiple cutaneous neoplasms in these patients. Key words: Antioxidant enzymes - Calcium transport Xeroderma pigmentosum
Xeroderma pigmentosum (XP) was first described by Hebra and Kaposi in 1874 [14]. The disorder represents a rare and heterogeneous group of diseases with an increased sensitivity to sunlight followed by a high frequency of sun-induced malignancies in the exposed skin Offprint requests to: K. U. Schallreuter
areas [14]. The typical clinical symptoms are a poikilodermatic skin with dyschromia, atrophy and telangiectasias with constant progression and development of multiple benign and malignant neoplasms. Most of these abnormalities arise from keratinocytes as precancerous lesions, as basal cell carcinoma and squamous cell carcinoma, whereas lentigo maligna arises from melanocytes and is the precursor for lentigo maligna melanoma. The overall frequency of this autosomal recessive disorder is approximately 1 in 250000, with a higher expression ( 1 0 - 2 5 in 100000) in Japan, Holland and Israel [14]. Two forms of XP have been discovered. In one form, a lack of those enzymes responsible for normal rates of unscheduled D N A synthesis occurs, and in the variant form, unscheduled DNA repair is normal, but the postreplicative maintenance of D N A integrity is absent [25]. In the excision-deficient group, there are nine complementation types ( A - I ) and each type is believed to have a distinct defect in D N A repair [2]. The majority of XP patients have the variant form of the disease and the complementation groups A, C, D, G and I are extremely rare [6, 7, 16]. Recently, it has been demonstrated that X-irradiated fibroblasts from XP heterozygotes yielded a two-fold increase in the frequency of chromatid breaks and gaps compared with control cells [16]. It has also been shown that XP cells are deficient in antioxidant defense against electrophiles generated by fluorescent light [24, 26], alkylating agents [13, 23] and, in some cases, ionizing radiation [4]. Very early studies of the epidermis of XP patients by electron microscopy showed significant membrane degeneration especially in mitocho~dria, Golgi apparatuses and other intracellular organelles. The exceptions were the melanosomes which appeared to be very resistant to UV-photochemical oxidative stress [22]. A study of 31 families of XP patients revealr that obligate heterozygotes in XP also have an increased predisposition to non-melanoma skin cancers compared with the normal population [35]. Therefore, even XP heterozygotes appear to be more susceptible to UV-mediated D N A damage.
450
K. U. Schallreuter et al.: Antioxidant defense and calcium transport in epidermis in xeroderma pigmentosum
The majority of molecular biology, genetic and biochemical studies on XP have been conducted with cultures of dermal fibroblasts. To date, there are only a few reports on keratinocytes and melanocytes which actually undergo oxidative stress and malignant conversions. Recently, the metabolic pathways for UV-generated oxygen radicals in the h u m a n epidermis have been reviewed [30]. Key enzymes for superoxide anion radical (O2) reduction have been identified as superoxide dismutase (SOD) and the thioredoxin reductase/thioredoxin (TR/T) electron transfer system [30]. SOD levels are low in the h u m a n epidermis c o m p a r e d with h u m a n liver and kidney [3]. This could be due to the conversion of 0 2 by SOD to hydrogen peroxide ( 0 2 - ) . This product is extremely toxic to cells in the epidermis through its UVphotochemical reaction yielding reactive hydroxyl radicals (OH-) [30]. OH. radicals are k n o w n to oxidize proteins, lipids and nucleic acids in a r a n d o m fashion [33]. The T R / T system in h u m a n keratinocytes represents 5% of the total acidic protein in the cell cytosol fraction [32]. The T R / T system (I) reduces O2 via 022- to water, and (II) regulates melanin biosynthesis [37]. This system is under allosteric regulation by calcium which binds to a single EF-hands site on T R to inhibit the electron transfer to oxidized thioredoxin (ToO [29, 32]. Therefore, the calcium status of the epidermis influences its oxidation status with high intracellular calcium leading to a decrease in antioxidant defense [32]. Two other enzymes play a m a j o r role as catalytic antioxidants in the h u m a n epidermis by metabolizing O22-. Catalase (CAT) converts 0 2- to molecular dioxygen, and the glutathione reductase/glutathione/glutathione peroxidase ( G R / G / G P ) system reduces O 2 - to water [30]. U n d e r normal conditions, the above four enzyme systems would provide adequate defense against O2 and 022-. However, an excess of UV-generated O2 deactivates catalase by irreversible oxidation of the four tetrapyrrole rings on the heme prosthetic group [1]. It is well established that some epidermis f r o m patients with XP contains higher than normal concentrations of hydrogen peroxide [36]. Keratinocyte cell cultures f r o m three XP patients revealed low catalase activities compared with healthy adult controls. Catalase was depressed even further in these ceils u p o n exposure to U V radiation. In addition, an increase in malignant conversion occurred [21]. Recently, it has been shown that T R is also deactivated approximately 50% by U V exposures below the minimal erythema dose ( M E D ) [34], whereas G R is mostly unaffected by U V A radiation. In experiments UVA-irradiated nude mouse skin, G R retained 85% of its control activities [12]. The aim of this study was to examine the redox status via CAT, SOD, T R and G R directly in the epidermis in h o m o z y g o u s and heterozygous XP patients.
England Nuclear, DuPont, Boston, Mass., USA. CAT, SOD and GR from human erythrocytes were purchased from Sigma Chemical Company, St. Louis, Mo., USA. TR and T were purified from human melanoma tissue by a modified method of Luthman and Holmgren [17, 28]. TR and T were resolved by FPLC on a mono Q HR 5/5 column in 0.05 M Tris-HC1 buffer, pH 7.5, using a 0-0.5 M NaC1 gradient. TR eluted at 0.12-0.13 M NaC1, calcium-bound TR (TRca2+) eluted at 0.10-0.105 M NaC1, and T at 0.3 M NaC1 (Fig. 1). 14CH3Hg incorporation into keratinocytes reduced thioredoxin (Tred) was measured according to the method of Schallreuter et al. [32]. Labelled calcium uptake experiments were performed on keratinocyte cell cultures grown in MCDB medium containing 0.1 x 10 -3 M calcium using the method of Schallreuter and Pittelkow [27]. Epidermal suction blisters were obtained from the XP family members and controls by a modified method of Pittelkow et al. [20]. Cell-free extracts were prepared from epidermal blister roofs by ultrasonication in 0.05 M Tris-HC1 buffer, pH 7.5, with a microprobe at the maximum setting (Heat Systems, Model 350 Ultrasonicator, New Brunswick, NJ, USA). Cell membranes were removed by centrifugation at 5000 rpm for 30 min at 0~ Protein concentrations were determined by the method of Kalb and Bernlohr [15]. An aliquot of 0.5 ml of extract was used for analysis by the mono Q HR 5/5 column equilibrated with 0.05 M Tris-HC1 buffer, pH 7.5, and a 0-0.5 M NaC1 gradient. Enzyme standards were chromatographed on FPLC under the conditions described above. SOD eluted at 0.075 M NaC1 and was assayed by ferricytochrome C reduction [11], CAT eluted at 0.16- 0.17 M NaC1 and was assayed by following oxygen evolution with a Clark oxygen electrode [8]. GR eluted at 0.22 M NaC1 and was assayed by the reduction of 5,5'-dithio-bis-2-nitrobenzoate (DTNB). The same method was used to assay TR [17, 28]. Membrane-associated TR was determined by nitroxide radical reduction [28, 31].
Patients and controls Family A. (Turkish, photo-skin type IV) (Fitzpatrick classification,
[10]). Father (35-years-old), mother (30-years-old), one healthy sister (10-years-old), one XP homozygote (13-years-old) (complementation group D). Case history. Boy (13-years-old) with normal physical and mental development, since age of 1 year, development of multiple freckles on sun-exposed areas; increasing numbers of basal cell carinoma and squamous cell carcinoma; no melanomas up to now; family history negative for XP. Family B. (German, photo-skin type II [10]). Mother (32-years-old),
one healthy sister (9-years-old), one XP homozygote (16-years-old) (complementation group C). Case history. Boy (16-years-old) with normal mental and physical development; since age of 2 years, increasing number of freckles on the total integument; family history negative for XP; first premalignant lesions at age 4, followed by basal cell carcinoma, squamous cell carcinoma, and at age 12, malignant melanoma; treatment with Tigason for 2.5 years without improvement in the frequency of basal cell carcinoma, squamous cell carcinoma or premalignant lesions. Controls. Six healthy probands with photo-skin type II (n = 3) and photo-skin type IV (n = 3) were used for the protocol.
Results Materials and methods Radiolabelled 14C-methyl mercuric chloride (42 mCi/mmol) was synthesized by the method of DeSimone et al. [9] and (radiolabelled 4%alcium chloride) (15mCi/mmol) was purchased from New
Table 1 summarizes a survey of the enzyme levels separated and identified f r o m epidermal blister roofs by using F P L C for b o t h families A and B. Figure 2 presents the
K. U. Schallreuteret al. : Antioxidantdefenseand calciumtransport in epidermis in xerodermapigmentosum 0.5
0.5 M NaCI
a
0.4
0.3
0.2
o.1
o.o 5
lO
15
20
25
30
0,5
0.5 M NaCI
b 0.4
E (CI
oo
0.3
o
0.2
t-
O
0.1
0.0 0
5
10
15
20
25
30
0.5
0.5M NaCI
C 0.4 -
0.3
0.2
4.0
9 !
!
3.0 o
I
z0~ "~
0.1
1.0
0.0 '~"
0
0.0
5
10
15
20
25
30
Fraction number, ml
Fig. l. a Purificationof melanoma thioredoxinreductase by FPLC (Aaso .m) full scale 1.0 A units on a mono Q column HR 5/5 in 0.05 M Tris buffer, pH 7.5, with a 0-0.5 M NaC1 gradient, This calcium-freeenzymeeluted at 0.12 M NaC1. b Purificationof 4SCalabelled melanomathioredoxin reductase (one 45Ca/TR molecule) on a mono Q columnin 0.05 M Tris buffer,pH 7.5. Calcium-bound thioredoxin reductase eluted at 0.105 M NaCI. e FPLC chromatography of 14CH3-HgC1labelledthioredoxinon a mono Q column in 0.05 M Tris buffer, pH 7.5 (A2so,~ full scale 1.0 A units). 14CH3HgCIlabelsthis proteinwitha 1: 1 stoichiometry( 9 -9 i.e.
/ 14CHaHgC1 + T
S~ ~4CH3-Hg-S.
\ SH
\ /
HS
T
451
individual FPLC data, in 0.05 M Tris-HC1 buffer, pH 7.5, for each member in family A using a 0 - 0 . 5 M NaC1 gradient. These results show considerable decreases in CAT, SOD and TR in both XP homozygotes compared with other family members and controls. Since TRca2 + can be distinguished from TR by FPLC, the levels of TRca~+ (inhibited enzyme) and TR (fully active enzyme) can be compared. As a consequence, TRca2+ has been used as a monitor for the cytosol calcium concentration at steady state in these samples [32]. Figure 3 presents the ratio TRc,2 . / T R in each individual family member. Both XP homozygotes indicate higher than normal intracellular calcium levels. For both mothers in families A and B, and for one daughter in family A, calciumlevels were high compared with control c skin. In order to test this calcium effect in more detail, keratinocytes were established from the XP homozygotes in families A and B, as well as from the mother in family B (obligate heterozygote), and the kinleticsl for 45Ca 2+ uptake were determined. Uptake rates and equilibria were compared with keratinocytes from a normal donor of skin type II. Figure 4 shows that both XP homozygotes maintain a high intracellular concentration~ of calcium at steady state, and the mother in family B (obligate heterozygote) had an intermediate level of~4SCa2+ between the skin type II control and the two XP homozygotes. FPLC analysis of keratinocyte cell extracts from the mother in family B afforded a separation of TRca2+ from TR in a ratio 5:3 (Fig. 5). This result is compatible with the analysis of the epidermal blister roof from the same patient yielding a TRca~+/TR of 2:1 (Table 1). Analysis of membrane-associated TR activities in 3 mm punch biopsies by electron spin resonance spectroscopy revealed that both XP homozygotes had significantly lower TR activities in sun-exposed skin versus unexposed skin compared with controls of similar skin type [31]. For XP patient A TR activity in sun-exposed skin was 30 units/3 mm biopsy per 10 rain and TR unexposed skin was 13 units/3 mm biopsy per 10 rain. The control skin activity for this XP patien t (skin type IV) was 20.4 _+ 3.0 (n = 6) [31]. For XP patient B TR unexposed skin was 3.5 units/3 mm biopsy per 10 min and TR sun-exposed skin was 2.5 units/3 mm biopsy per 10 min. The control skin activity for this XP patient (skin type II) was 14.6 _+ 3.0 (n = 7) [31]. (It should be noted that for patients with lower skin types (I and II), the membrane-associated TR assay has a greater standard deviation owing to lower enzyme activities.) The influence of UV radiation on the expression of intracellular TR activity in XP was evaluated further by analyses of the intracellular concentration of T~od in keratinocytes established from sun-exposed versus unexposed skin ofXP homozygote B. Table 2 shows that TR activity is significantly depressed ( - 5 0 % ) in keratinocytes from sun-exposed skin of this XP hon:tozygote compared with his unexposed skin and with cells from his mother (obligate heterozygote) and his sister. The low concentration of Trea in keratinocytes from sun-exposed skin in XP confirms local variations with more oxidizing intracellular conditions.
452
K . U . Schallreuter et al.: Antioxidant defense and calcium transport in epidermis in xeroderma pigmentosum
Table 1. FPLC separation and identification of enzymes involved in 0 2 and 022 reduction from epidermal blisler roofs taken from families A and B with XP. Enzyme levels are expressed as gg protein/rag epidermal blister-derived celt extract Subject
SOD
CAT
GR
TRca2 +
TR
Family A (XP-complementation group D): Father Mother Daugtlzer Son (XP) Type IV skin (control) mean (n = 5)
6.53 5.25 656 1.6 6.2 _+ 0.5
12.45 12.25 14+05 8.0 12.5 _+ 1.2
3.06 5.0 n_ d. 4.8 4.7 _+ 0.4
5.1 4.0 4.2 2.4 5.60 _+ 0.2
4.7 2.5 2.16 1.6 5.1 _+ 0.75
Family B (XP-complementation group C): Mother Son (XP) Type II skin (control mean (n = 5)
4.1 2.0 3.8 + 0.35
10.5 1.4 12.1 _+ 0.8
3.6 3.9 3.7 _+ 0.4
5.0 2.6 4.6 -t- 0.3
2.5 1.5 3.70 _+ 0.80
b1
tt II
003 4!
0.03
mc,,~ CATI I
- 0,5M NaCL
0.5M NaCI 0.02-
0.02 -
I
',j m(
0.01 000 o,I U.l 0 Z
0
~i
o
1=5
10
soDIT. I
I
~,
J ,
2=5
0
3~3
u,J/t,.., o
;
;o
1'~
2b
g
30
d
I I
II II II II II
| I l I SOD
; TRc2. CAT I 0,02-
"l IT. Ii
0.01-
0
2=0
I I I
0.03-
GR
0.01
I
