APMIS 122: 880–889
© 2014 APMIS. Published by John Wiley & Sons Ltd. DOI 10.1111/apm.12251
Expression of cancer-related carbonic anhydrases IX and XII in normal skin and skin neoplasms 1 € LEO SYRJANEN, TIINA LUUKKAALA,2 MARI LEPPILAMPI,3 MATTI KALLIOINEN,4 SILVIA PASTOREKOVA,5 JAROMIR PASTOREK,5 ABDUL WAHEED,6 WILLIAM S. SLY,6 SEPPO PARKKILA1,7 and TUOMO KARTTUNEN4
Institute of Biomedical Technology and School of Medicine, University of Tampere, Tampere; 2Pirkanmaa Hospital District and School of Health Sciences, Science Center, University of Tampere, Tampere; 3 Department of Clinical Chemistry, Oulu University Hospital, Oulu; 4Department of Pathology, University of Oulu and Oulu University Hospital, Medical Research Center, Oulu, Finland; 5Department of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, Bratislava, Slovak Republic; 6Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO, USA; 7Fimlab Laboratories and Tampere University Hospital, Tampere, Finland 1
Leo Syrj€anen, Tiina Luukkaala, Mari Leppilampi, Matti Kallioinen, Silvia Pastorekova, Jaromir Pastorek, Abdul Waheed, William S. Sly, Seppo Parkkila, Tuomo Karttunen. Expression of cancer-related carbonic anhydrases IX and XII in normal skin and skin neoplasms. APMIS 2014; 122: 880–889. Purpose of the study was to evaluate the presence of hypoxia-inducible, tumour-associated carbonic anhydrases IX and XII in normal skin and a series of cutaneous tumours. Human tumour samples were taken during surgical operations performed on 245 patients and were immunohistochemically stained. A histological score value was calculated for statistical analyses which were performed using SPSS for Windows, versions 17.0 and 20.0. In normal skin, the highest expression of CA IX was detected in hair follicles, sebaceous glands, and basal parts of epidermis. CA XII was detected in all epithelial components of skin. Both CA IX and CA XII expression levels were significantly different in epidermal, appendigeal, and melanocytic tumour categories. Both CA IX and XII showed the most intense immunostaining in epidermal tumours, whereas virtually all melanocytic tumours were devoid of CA IX and XII immunostaining. In premalignant lesions, CA IX expression significantly increased when the tumours progressed to more severe dysplasia forms. Both CA IX and XII are highly expressed in different epithelial components of skin. They are also highly expressed in epidermal tumours, in which CA IX expression levels also correlate with the dysplasia grade. Interestingly, both isozymes are absent in melanocytic tumours. Key words: CA IX; CA XI; skin; skin neoplasm. Leo Syrj€anen, School of Medicine, University of Tampere, Medisiinarinkatu 3, Tampere 33014, Finland. e-mail: leo.syrjanen@uta.fi
Carbonic anhydrases (CAs) are widespread metalloenzymes that catalyse the reversible hydration of carbon dioxide into bicarbonate ion and proton according to the following reaction: CO2 + H2O ↔ H+ + HCO3 (1). The main function of CAs is the maintaining of acid–base balance but they are involved in many other reactions like bone resorption and many biosynthetic processes (2). The mammalian a-CA family consists of 13 active enzymes of which 12 are found in humans (3). CAs I, II, III, VII, and XIII are cytosolic enzymes (2, 4, 5), CAs IV, IX, XII, XIV, and XV (which is not Received 14 October 2013. Accepted 20 December 2013
880
expressed in humans) are membrane-bound (6–11), CAs VA and VB are found in mitochondria, (12) and CA VI is the only secreted form (13). Generally CAs have a high catalytic activity but there are differences between these enzymes in both efficiency and inhibitory properties (1). CAs IX and XII are hypoxia-inducible, tumour-associated members of the human a-CA family. CA IX shows only limited expression in normal tissues but its expression is highly elevated in various cancers like colorectal and lung carcinomas (14, 15). The cDNA coding for human CA IX was originally cloned in 1994 and the CA9 gene was further characterized in 1996 (16, 17). Recently,
CAS IX AND XII IN NORMAL SKIN AND TUMOURS
biochemical characterization of CA IX protein revealed that it is one of the most active CAs (3). The crystal structure of CA IX catalytic domain was reported in the year 2009 (18), and it showed a unique quaternary structure among the a-CA enzymes. Monomeric CA IX is a 422 amino acid glycoprotein and consists of four different domains: an N-terminal proteoglycan-related domain, CA domain, transmembrane domain, and short intracytoplasmic tail. CA XII was identified and characterized independently by two research groups in 1998 (9, 19). After removal of the signal sequence, the enzyme contains 325 amino acids and possesses a CA domain, an additional short extracellular part, a hydrophobic transmembrane domain and a C-terminal cytoplasmic tail. The expression of CA XII shows some similarities with CA IX. Both isozymes are induced by hypoxia and are located at the basolateral plasma membranes. They have quite limited expression in normal tissues but show increased expression in tumour cell lines and certain cancers. Recently, it was shown that these CAs are expressed in many foetal tissues during human development (20). There is only a limited amount of published data on the expression of CAs IX and XII in the human skin. CA IX has been reported in hair follicles and sebaceous glands of normal skin, while epithelial cells, capillaries, and sweat glands have been negative (14, 20, 21). CA XII has been demonstrated in basal cells of epidermis and sweat glands of the skin (14). The aim of this study was to examine the expression of cancer-related CAs IX and XII in the normal human skin and various cutaneous tumours. Although skin cancers are clinically important neoplasms, they have not been extensively studied for the expression of these CA isozymes.
MATERIALS AND METHODS Preparation of tissues Human tumour samples were taken during surgical operations performed on 245 patients at Oulu University Hospital. The study was approved by the Research Ethics Committee of Oulu University Hospital and permission for sample collection was also obtained from the National Authority for Medicolegal Affairs (Helsinki, Finland). The sample material consisted of: 61 skin appendigeal tumours, 21 basal cell carcinomas, 10 basosquamous carcinomas, 63 melanocytic tumours of which 31 were benign and 32 malignant melanomas, 10 cases of mycosis fungoides, 22 squamous cell acanthomas, and 61 squamous cell carcinomas or squamous cell dysplasias. The normal skin was examined from these same samples, however avoiding regions close to lesion. The specimens were fixed in © 2014 APMIS. Published by John Wiley & Sons Ltd
buffered 4% formaldehyde. The samples were then dehydrated, embedded in paraffin in a vacuum oven at +58 °C. Newly made 5-lm sections were placed on gelatin-coated microscope slides. Routine haematoxylin and eosin (H&E) staining was carried out after tissue preparation.
Antibodies and immunohistochemical staining procedure The monoclonal antibody M75, recognizing the N-terminal domain of human CA IX, has been described previously (22). The rabbit anti-human CA XII antiserum against truncated, secretory form of CA XII has been characterized by Karhumaa et al. (23) Normal rabbit serum was used for control staining. Immunohistochemical staining was performed using automated Lab Vision Autostainer 480 (ImmunoVision Technologies Co., Brisbane, CA, USA) and polymer based Power Vision+TM Poly-HRP IHC Kit reagents (ImmunoVision Technologies, Co.). Immunostaining was performed according to the following protocol: (i) rinsing in wash buffer; (ii) treatment in 3% H2O2 in ddH2O for 5 min and rinsing in wash buffer; (iii) blocking with Universal IHC Blocking/Diluent for 30 min and rinsing in wash buffer; (iv) incubation with the primary antibody or NRS diluted 1:1000 (anti-CA IX) or 1:2000 (anti-CA XII and NRS) in Universal IHC Blocking/Diluent for 30 min; (v) rinsing in wash buffer for 3 9 5 min; (vi) incubation in Poly-HRP-conjugated anti-mouse or anti-Rabbit IgG for 30 min and rinsing in wash buffer for 3 9 5 min; (vii) incubation in DAB (3,3′–diaminobenzidine tetrahydrochloride) solution (one drop DAB solution A and one drop DAB solution B with 1 ml) ddH2O for 6 min; (viii) rinsing with ddH2O; (ix) CuSO4 treatment for 5 min to enhance the signal; and (x) rinsing with ddH2O. All procedures were carried out at room temperature. All specimens were analysed by investigators (ML, TK), who assessed cases simultaneously. A consensus score for each specimen was obtained and registered. In addition, a subset of cases was similarly assessed with a third assessor (MK). Both the proportion of stained cells (0–100%) and staining intensity (0, no staining; 1, weak reaction; 2, moderate reaction; 3, strong reaction) were examined. Figure 1A–F show examples of different staining intensities. Intensity was assessed according to predominant positive expression intensity in each anatomical structure or lesion type. For statistical analysis, the proportion of stained cells and intensity were converted to staining reactivity values according to the following equations. For CA IX-stained epidermis: (3 9 proportion of epidermal thickness showing expression (%) 9 proportion epidermal lateral extent showing expression (%)/10000) + intensity. For the other tissue components: (3 9 proportion of stained cells (%)/100) + intensity. Using these equations, both the number of stained cells and staining intensity can be evaluated on a scale 0–3. By summing these values, we obtained the histological score values that ranged from 0 (minimum) to 6 (maximum).
Statistical analyses For comparison of the expression of CA IX and CA XII staining reactivity between groups representing gender, light exposure, benign and malignant lesions, tumourinvasiveness, and the difference between solar keratosis
881
€ SYRJANEN et al.
A
B
C
D
E
F
Fig. 1. Examples of CA IX and CA XII expression in normal skin and in epidermal dysplasia. In normal skin, expression of CA IX is present in the basal parts of epidermis (A) and in the outer root cells of the hair follicle (B). Strong expression of CA IX marks epidermal dysplasia (solar keratosis; C). In squamous carcinoma moderate expression is present in the invasive front region (D). Expression of CA XII is seen in the basal parts of epidermis (E) and in the sebaceal and sweat glands (F). In the figures, examples of weak expression levels have been marked with one arrowhead, moderate with two adjacent arrowheads, and strong with arrows. and Bowen’s disease, we used Mann–Whitney test. Expression levels of CA IX and CA XII were analysed by Median test due to the different shapes of distributions in the analyses of all preinvasive epidermal tumours which were categorized according to dysplasia (slight, moderate, severe carcinoma in situ). Correlations of epithelial staining reactions in the epidermal lesion with age and expressions in adjacent healthy epidermis were assessed by Spearmann’s correlations (rho). Statistical analyses were performed by SPSS for Windows, versions 17.0 and 20.0. p-values (p) under 0.05 were considered as statistical significant.
RESULTS AND DISCUSSION CA IX is a unique CA isozyme in many respects including its structure and distribution. Although it is present in many tumours, it is usually absent in normal tissues from where the tumour has originated. In the present study, we found that CA 882
IX is moderately expressed in the normal skin and that the other cancer-associated isozyme, CA XII, showed even more intense immunostaining. Examples of CA IX and CA XII expression in normal skin and in epidermal dysplasia can be seen in Fig. 1. The expression of CA IX and XII varied substantially between different parts of the normal skin (Fig. 2). The sweat gland was the only epithelial part of the skin showing no expression of CA IX. The highest signal for CA IX was found in the hair follicles and sebaceous glands. The epidermis also showed substantial expression, which was mostly present in the basal parts and seemed to have a preferential location around hair follicle openings. Obviously, the role of CA IX in the physiology of hair follicles and in the sebum formation warrants further studies. CA XII was detected in all epithelial components of the normal skin. The maximum staining reactivity values for CA XII © 2014 APMIS. Published by John Wiley & Sons Ltd
CAS IX AND XII IN NORMAL SKIN AND TUMOURS
Fig. 2. Expression of CA IX and CA XII in the normal skin structures examined in 32 and 49 specimens, respectively. Distribution of values was shown by medians (black line), interquartile ranges (box), and ranges (line bar). Outliers and extreme cases were expressed as dots or stars.
were 6 on the scale of 0–6. Marked expression of CA XII was observed in the sebaceous glands and epidermis, and moderate expression was seen in the hair follicles. Especially striking was the very high expression of CA XII in the sweat glands which may implicate a role for this enzyme in perspiration. It has been shown that E143K mutation in CAXII causes imbalance in chloride homeostasis in sweat glands (24). The healthy tissues were divided into two groups depending on the exposure to the sunlight in that region. For example, the facial skin was placed to © 2014 APMIS. Published by John Wiley & Sons Ltd
the ‘sunlight exposure’ group and skin of the knee was in the ‘non-sunlight exposure’ group. It was shown that the expression levels of both CA IX and CA XII were significantly increased in the regions exposed to sunlight, p-values being
© 2014 APMIS. Published by John Wiley & Sons Ltd. DOI 10.1111/apm.12251
Expression of cancer-related carbonic anhydrases IX and XII in normal skin and skin neoplasms 1 € LEO SYRJANEN, TIINA LUUKKAALA,2 MARI LEPPILAMPI,3 MATTI KALLIOINEN,4 SILVIA PASTOREKOVA,5 JAROMIR PASTOREK,5 ABDUL WAHEED,6 WILLIAM S. SLY,6 SEPPO PARKKILA1,7 and TUOMO KARTTUNEN4
Institute of Biomedical Technology and School of Medicine, University of Tampere, Tampere; 2Pirkanmaa Hospital District and School of Health Sciences, Science Center, University of Tampere, Tampere; 3 Department of Clinical Chemistry, Oulu University Hospital, Oulu; 4Department of Pathology, University of Oulu and Oulu University Hospital, Medical Research Center, Oulu, Finland; 5Department of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, Bratislava, Slovak Republic; 6Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO, USA; 7Fimlab Laboratories and Tampere University Hospital, Tampere, Finland 1
Leo Syrj€anen, Tiina Luukkaala, Mari Leppilampi, Matti Kallioinen, Silvia Pastorekova, Jaromir Pastorek, Abdul Waheed, William S. Sly, Seppo Parkkila, Tuomo Karttunen. Expression of cancer-related carbonic anhydrases IX and XII in normal skin and skin neoplasms. APMIS 2014; 122: 880–889. Purpose of the study was to evaluate the presence of hypoxia-inducible, tumour-associated carbonic anhydrases IX and XII in normal skin and a series of cutaneous tumours. Human tumour samples were taken during surgical operations performed on 245 patients and were immunohistochemically stained. A histological score value was calculated for statistical analyses which were performed using SPSS for Windows, versions 17.0 and 20.0. In normal skin, the highest expression of CA IX was detected in hair follicles, sebaceous glands, and basal parts of epidermis. CA XII was detected in all epithelial components of skin. Both CA IX and CA XII expression levels were significantly different in epidermal, appendigeal, and melanocytic tumour categories. Both CA IX and XII showed the most intense immunostaining in epidermal tumours, whereas virtually all melanocytic tumours were devoid of CA IX and XII immunostaining. In premalignant lesions, CA IX expression significantly increased when the tumours progressed to more severe dysplasia forms. Both CA IX and XII are highly expressed in different epithelial components of skin. They are also highly expressed in epidermal tumours, in which CA IX expression levels also correlate with the dysplasia grade. Interestingly, both isozymes are absent in melanocytic tumours. Key words: CA IX; CA XI; skin; skin neoplasm. Leo Syrj€anen, School of Medicine, University of Tampere, Medisiinarinkatu 3, Tampere 33014, Finland. e-mail: leo.syrjanen@uta.fi
Carbonic anhydrases (CAs) are widespread metalloenzymes that catalyse the reversible hydration of carbon dioxide into bicarbonate ion and proton according to the following reaction: CO2 + H2O ↔ H+ + HCO3 (1). The main function of CAs is the maintaining of acid–base balance but they are involved in many other reactions like bone resorption and many biosynthetic processes (2). The mammalian a-CA family consists of 13 active enzymes of which 12 are found in humans (3). CAs I, II, III, VII, and XIII are cytosolic enzymes (2, 4, 5), CAs IV, IX, XII, XIV, and XV (which is not Received 14 October 2013. Accepted 20 December 2013
880
expressed in humans) are membrane-bound (6–11), CAs VA and VB are found in mitochondria, (12) and CA VI is the only secreted form (13). Generally CAs have a high catalytic activity but there are differences between these enzymes in both efficiency and inhibitory properties (1). CAs IX and XII are hypoxia-inducible, tumour-associated members of the human a-CA family. CA IX shows only limited expression in normal tissues but its expression is highly elevated in various cancers like colorectal and lung carcinomas (14, 15). The cDNA coding for human CA IX was originally cloned in 1994 and the CA9 gene was further characterized in 1996 (16, 17). Recently,
CAS IX AND XII IN NORMAL SKIN AND TUMOURS
biochemical characterization of CA IX protein revealed that it is one of the most active CAs (3). The crystal structure of CA IX catalytic domain was reported in the year 2009 (18), and it showed a unique quaternary structure among the a-CA enzymes. Monomeric CA IX is a 422 amino acid glycoprotein and consists of four different domains: an N-terminal proteoglycan-related domain, CA domain, transmembrane domain, and short intracytoplasmic tail. CA XII was identified and characterized independently by two research groups in 1998 (9, 19). After removal of the signal sequence, the enzyme contains 325 amino acids and possesses a CA domain, an additional short extracellular part, a hydrophobic transmembrane domain and a C-terminal cytoplasmic tail. The expression of CA XII shows some similarities with CA IX. Both isozymes are induced by hypoxia and are located at the basolateral plasma membranes. They have quite limited expression in normal tissues but show increased expression in tumour cell lines and certain cancers. Recently, it was shown that these CAs are expressed in many foetal tissues during human development (20). There is only a limited amount of published data on the expression of CAs IX and XII in the human skin. CA IX has been reported in hair follicles and sebaceous glands of normal skin, while epithelial cells, capillaries, and sweat glands have been negative (14, 20, 21). CA XII has been demonstrated in basal cells of epidermis and sweat glands of the skin (14). The aim of this study was to examine the expression of cancer-related CAs IX and XII in the normal human skin and various cutaneous tumours. Although skin cancers are clinically important neoplasms, they have not been extensively studied for the expression of these CA isozymes.
MATERIALS AND METHODS Preparation of tissues Human tumour samples were taken during surgical operations performed on 245 patients at Oulu University Hospital. The study was approved by the Research Ethics Committee of Oulu University Hospital and permission for sample collection was also obtained from the National Authority for Medicolegal Affairs (Helsinki, Finland). The sample material consisted of: 61 skin appendigeal tumours, 21 basal cell carcinomas, 10 basosquamous carcinomas, 63 melanocytic tumours of which 31 were benign and 32 malignant melanomas, 10 cases of mycosis fungoides, 22 squamous cell acanthomas, and 61 squamous cell carcinomas or squamous cell dysplasias. The normal skin was examined from these same samples, however avoiding regions close to lesion. The specimens were fixed in © 2014 APMIS. Published by John Wiley & Sons Ltd
buffered 4% formaldehyde. The samples were then dehydrated, embedded in paraffin in a vacuum oven at +58 °C. Newly made 5-lm sections were placed on gelatin-coated microscope slides. Routine haematoxylin and eosin (H&E) staining was carried out after tissue preparation.
Antibodies and immunohistochemical staining procedure The monoclonal antibody M75, recognizing the N-terminal domain of human CA IX, has been described previously (22). The rabbit anti-human CA XII antiserum against truncated, secretory form of CA XII has been characterized by Karhumaa et al. (23) Normal rabbit serum was used for control staining. Immunohistochemical staining was performed using automated Lab Vision Autostainer 480 (ImmunoVision Technologies Co., Brisbane, CA, USA) and polymer based Power Vision+TM Poly-HRP IHC Kit reagents (ImmunoVision Technologies, Co.). Immunostaining was performed according to the following protocol: (i) rinsing in wash buffer; (ii) treatment in 3% H2O2 in ddH2O for 5 min and rinsing in wash buffer; (iii) blocking with Universal IHC Blocking/Diluent for 30 min and rinsing in wash buffer; (iv) incubation with the primary antibody or NRS diluted 1:1000 (anti-CA IX) or 1:2000 (anti-CA XII and NRS) in Universal IHC Blocking/Diluent for 30 min; (v) rinsing in wash buffer for 3 9 5 min; (vi) incubation in Poly-HRP-conjugated anti-mouse or anti-Rabbit IgG for 30 min and rinsing in wash buffer for 3 9 5 min; (vii) incubation in DAB (3,3′–diaminobenzidine tetrahydrochloride) solution (one drop DAB solution A and one drop DAB solution B with 1 ml) ddH2O for 6 min; (viii) rinsing with ddH2O; (ix) CuSO4 treatment for 5 min to enhance the signal; and (x) rinsing with ddH2O. All procedures were carried out at room temperature. All specimens were analysed by investigators (ML, TK), who assessed cases simultaneously. A consensus score for each specimen was obtained and registered. In addition, a subset of cases was similarly assessed with a third assessor (MK). Both the proportion of stained cells (0–100%) and staining intensity (0, no staining; 1, weak reaction; 2, moderate reaction; 3, strong reaction) were examined. Figure 1A–F show examples of different staining intensities. Intensity was assessed according to predominant positive expression intensity in each anatomical structure or lesion type. For statistical analysis, the proportion of stained cells and intensity were converted to staining reactivity values according to the following equations. For CA IX-stained epidermis: (3 9 proportion of epidermal thickness showing expression (%) 9 proportion epidermal lateral extent showing expression (%)/10000) + intensity. For the other tissue components: (3 9 proportion of stained cells (%)/100) + intensity. Using these equations, both the number of stained cells and staining intensity can be evaluated on a scale 0–3. By summing these values, we obtained the histological score values that ranged from 0 (minimum) to 6 (maximum).
Statistical analyses For comparison of the expression of CA IX and CA XII staining reactivity between groups representing gender, light exposure, benign and malignant lesions, tumourinvasiveness, and the difference between solar keratosis
881
€ SYRJANEN et al.
A
B
C
D
E
F
Fig. 1. Examples of CA IX and CA XII expression in normal skin and in epidermal dysplasia. In normal skin, expression of CA IX is present in the basal parts of epidermis (A) and in the outer root cells of the hair follicle (B). Strong expression of CA IX marks epidermal dysplasia (solar keratosis; C). In squamous carcinoma moderate expression is present in the invasive front region (D). Expression of CA XII is seen in the basal parts of epidermis (E) and in the sebaceal and sweat glands (F). In the figures, examples of weak expression levels have been marked with one arrowhead, moderate with two adjacent arrowheads, and strong with arrows. and Bowen’s disease, we used Mann–Whitney test. Expression levels of CA IX and CA XII were analysed by Median test due to the different shapes of distributions in the analyses of all preinvasive epidermal tumours which were categorized according to dysplasia (slight, moderate, severe carcinoma in situ). Correlations of epithelial staining reactions in the epidermal lesion with age and expressions in adjacent healthy epidermis were assessed by Spearmann’s correlations (rho). Statistical analyses were performed by SPSS for Windows, versions 17.0 and 20.0. p-values (p) under 0.05 were considered as statistical significant.
RESULTS AND DISCUSSION CA IX is a unique CA isozyme in many respects including its structure and distribution. Although it is present in many tumours, it is usually absent in normal tissues from where the tumour has originated. In the present study, we found that CA 882
IX is moderately expressed in the normal skin and that the other cancer-associated isozyme, CA XII, showed even more intense immunostaining. Examples of CA IX and CA XII expression in normal skin and in epidermal dysplasia can be seen in Fig. 1. The expression of CA IX and XII varied substantially between different parts of the normal skin (Fig. 2). The sweat gland was the only epithelial part of the skin showing no expression of CA IX. The highest signal for CA IX was found in the hair follicles and sebaceous glands. The epidermis also showed substantial expression, which was mostly present in the basal parts and seemed to have a preferential location around hair follicle openings. Obviously, the role of CA IX in the physiology of hair follicles and in the sebum formation warrants further studies. CA XII was detected in all epithelial components of the normal skin. The maximum staining reactivity values for CA XII © 2014 APMIS. Published by John Wiley & Sons Ltd
CAS IX AND XII IN NORMAL SKIN AND TUMOURS
Fig. 2. Expression of CA IX and CA XII in the normal skin structures examined in 32 and 49 specimens, respectively. Distribution of values was shown by medians (black line), interquartile ranges (box), and ranges (line bar). Outliers and extreme cases were expressed as dots or stars.
were 6 on the scale of 0–6. Marked expression of CA XII was observed in the sebaceous glands and epidermis, and moderate expression was seen in the hair follicles. Especially striking was the very high expression of CA XII in the sweat glands which may implicate a role for this enzyme in perspiration. It has been shown that E143K mutation in CAXII causes imbalance in chloride homeostasis in sweat glands (24). The healthy tissues were divided into two groups depending on the exposure to the sunlight in that region. For example, the facial skin was placed to © 2014 APMIS. Published by John Wiley & Sons Ltd
the ‘sunlight exposure’ group and skin of the knee was in the ‘non-sunlight exposure’ group. It was shown that the expression levels of both CA IX and CA XII were significantly increased in the regions exposed to sunlight, p-values being
