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Inhibition of skin tumor growth and angiogenesis in vivo by activation of cannabinoid receptors
M. Llanos Casanova,1 Cristina Blázquez,2 Jesús Martínez-Palacio,1 Concepción Villanueva,3 M. Jesús Fernández-Aceñero,3 John W. Huffman,4 José L. Jorcano,1 and Manuel Guzmán2
1Project on Cellular and Molecular Biology and Gene Therapy, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas, Madrid, Spain2 Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, Madrid, Spain3 Department of Pathology, Hospital General de Móstoles, Madrid, Spain4 Department of Chemistry, Clemson University, Clemson, South Carolina, USA
Address correspondence to: Manuel Guzmán, Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, 28040 Madrid, Spain. Phone: 34-913944668; Fax: 34-913944672; E-mail: mgp@bbm1.ucm.es.
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1Project on Cellular and Molecular Biology and Gene Therapy, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas, Madrid, Spain2 Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, Madrid, Spain3 Department of Pathology, Hospital General de Móstoles, Madrid, Spain4 Department of Chemistry, Clemson University, Clemson, South Carolina, USA
Address correspondence to: Manuel Guzmán, Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, 28040 Madrid, Spain. Phone: 34-913944668; Fax: 34-913944672; E-mail: mgp@bbm1.ucm.es.
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1Project on Cellular and Molecular Biology and Gene Therapy, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas, Madrid, Spain2 Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, Madrid, Spain3 Department of Pathology, Hospital General de Móstoles, Madrid, Spain4 Department of Chemistry, Clemson University, Clemson, South Carolina, USA
Address correspondence to: Manuel Guzmán, Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, 28040 Madrid, Spain. Phone: 34-913944668; Fax: 34-913944672; E-mail: mgp@bbm1.ucm.es.
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1Project on Cellular and Molecular Biology and Gene Therapy, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas, Madrid, Spain2 Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, Madrid, Spain3 Department of Pathology, Hospital General de Móstoles, Madrid, Spain4 Department of Chemistry, Clemson University, Clemson, South Carolina, USA
Address correspondence to: Manuel Guzmán, Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, 28040 Madrid, Spain. Phone: 34-913944668; Fax: 34-913944672; E-mail: mgp@bbm1.ucm.es.
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1Project on Cellular and Molecular Biology and Gene Therapy, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas, Madrid, Spain2 Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, Madrid, Spain3 Department of Pathology, Hospital General de Móstoles, Madrid, Spain4 Department of Chemistry, Clemson University, Clemson, South Carolina, USA
Address correspondence to: Manuel Guzmán, Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, 28040 Madrid, Spain. Phone: 34-913944668; Fax: 34-913944672; E-mail: mgp@bbm1.ucm.es.
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1Project on Cellular and Molecular Biology and Gene Therapy, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas, Madrid, Spain2 Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, Madrid, Spain3 Department of Pathology, Hospital General de Móstoles, Madrid, Spain4 Department of Chemistry, Clemson University, Clemson, South Carolina, USA
Address correspondence to: Manuel Guzmán, Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, 28040 Madrid, Spain. Phone: 34-913944668; Fax: 34-913944672; E-mail: mgp@bbm1.ucm.es.
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1Project on Cellular and Molecular Biology and Gene Therapy, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas, Madrid, Spain2 Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, Madrid, Spain3 Department of Pathology, Hospital General de Móstoles, Madrid, Spain4 Department of Chemistry, Clemson University, Clemson, South Carolina, USA
Address correspondence to: Manuel Guzmán, Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, 28040 Madrid, Spain. Phone: 34-913944668; Fax: 34-913944672; E-mail: mgp@bbm1.ucm.es.
Find articles by Jorcano, J. in: JCI | PubMed | Google Scholar
1Project on Cellular and Molecular Biology and Gene Therapy, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas, Madrid, Spain2 Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, Madrid, Spain3 Department of Pathology, Hospital General de Móstoles, Madrid, Spain4 Department of Chemistry, Clemson University, Clemson, South Carolina, USA
Address correspondence to: Manuel Guzmán, Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, 28040 Madrid, Spain. Phone: 34-913944668; Fax: 34-913944672; E-mail: mgp@bbm1.ucm.es.
Find articles by Guzmán, M. in: JCI | PubMed | Google Scholar
Published January 1, 2003 - More info
J Clin Invest. 2003;111(1):43–50. https://doi.org/10.1172/JCI16116.
© 2003 The American Society for Clinical Investigation
Received: June 7, 2002; Accepted: November 19, 2002
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Methods
Cannabinoids. JWH-133 was prepared in J.W. Huffman’s laboratory (25). WIN-55,212-2 was from Sigma-Aldrich (St. Louis, Missouri, USA). SR141716 and SR144528 were kindly given by Sanofi-Synthelabo (Montpellier, France).
Cell culture. The mouse tumorigenic epidermal cell lines PDV.C57 and HaCa4, and the nontransformed epidermal cell lines MCA3D (mouse) and HaCat (human), were routinely maintained in DMEM supplemented with 10% FCS. Twenty-four hours before the experiments, cells were transferred to low serum (0.5%) DMEM. Primary human keratinocytes (Biowhittaker Europe SPRL, Vervier, Belgium) were grown and cultured for the experiments in KGM-2 medium (Biowhittaker Europe SPRL). Stock solutions of cannabinoid ligands were prepared in DMSO. Control incubations had the corresponding DMSO content. No significant influence of DMSO was observed on cell viability at the final concentration used (0.1–0.2%, vol/vol).
Human tumor samples. Formalin-fixed, paraffin-embedded, human tumor samples were obtained from the files of the Department of Pathology, Hospital General de Móstoles (Madrid, Spain). Six-micrometer tumor sections were stained with either hematoxylin-eosin or used for immunohistochemistry (see below). The normal skin sample (Figures 1 and 3, skin) came from the face of a 70-year-old man. The slowly growing tumor sample (Figure 3, BCC) came from the face of a 69-year-old woman. The histological analysis revealed the growth of basaloid cell solid nests at the dermoepidermal junction with diagnostic characteristics of basal cell carcinoma. The highly malignant tumor sample (Figures 1 and 3, SCC) came from the retroauricular skin of a 73-year-old man. The histological analysis showed the neoplastic growth of epithelial cells, with squamous differentiation and focal areas of acantholysis.
Figure 1Western blot analysis of cannabinoid receptor expression in normal skin and skin tumors. (a) CB1 and CB2 receptor expression in murine and human epidermal cell lines, normal skin, and skin tumors. (b) Controls with the anti-CB1 or anti-CB2 Ab-blocking peptide are shown (see Methods and Results for explanation). Mouse papillomas were generated by chemical carcinogenesis (as described in ref. 9), while mouse squamous cell carcinomas (SCCs) were generated by inoculation of PDV.C57 epidermal tumor cells as described in Methods. The source of human samples is described in Methods. Images of representative samples are shown. Similar results were obtained in at least two other blots. PB, immortalized mouse nontumorigenic cell line derived from SENCAR mice papillomas; HK, human keratinocytes; m, mouse; h, human.
Figure 3Immunohistochemical analysis of cannabinoid receptor expression in human normal skin and skin tumors. (a) Immunolocalization of CB1 and CB2 receptors. (b) Controls without primary Ab (only with secondary biotinylated anti-rabbit Ab), as well as controls with the Ab-blocking peptides, are shown (see Methods and Results for explanation). The source of samples is described in Methods. BCC, basal cell carcinoma; SCC, squamous cell carcinoma; Hf, hair follicle.
Tumor induction in mice. Malignant skin tumors were induced in nude (NMRI nu) mice by subcutaneous flank inoculation of 106 PDV.C57 epidermal tumor cells. When tumors had reached an average volume of 500 mm3 (range, 400–600 mm3), a continuous flow pump (Alzet 2002; Alza Corporation, Palo Alto, California, USA) was surgically implanted on the flank of every mouse near the site of tumor inoculation. The pump was filled with either vehicle, 1,580 μg of WIN-55,212-2, or 1,580 μg of JWH-133 in 236 μl PBS supplemented with 5 mg/ml BSA and operated at a flux of 0.52 μl/h for 11 days. Tumors were measured with an external caliper and their volume calculated as (4π/3) × (width/2)2 × (length/2). Cannabinoid treatment did not affect animal weight gain (data not shown).
Viability and apoptosis in vitro. Cell viability in the cultures was determined by the 3-4,5-dimethylthiazol-2,5-diphenyltetrazolium bromide thiazol blue test. Apoptosis was determined by both TUNEL staining (21) and oligonucleosomal DNA fragmentation (26) according to kit manufacturer’s instructions (Boehringer Mannheim GmbH, Mannheim, Germany).
Proliferation and apoptosis in vivo. For proliferation assays, mice received an intraperitoneal injection of BrdU (120 mg/kg body weight) (Boehringer Mannheim GmbH) 2 hours before tumor harvesting. Detection of BrdU-positive cells was performed using an anti-BrdU mouse mAb (Boehringer Mannheim GmbH) as described (9). Apoptosis was determined by TUNEL staining (Boehringer Mannheim GmbH) according to kit manufacturer’s instructions (21).
Western blot analysis. Particulate cell or tissue fractions were subjected to SDS-PAGE, and proteins were transferred from the gels onto polyvinylidene fluoride membranes. To determine cannabinoid receptor expression the blots were incubated with polyclonal Ab’s raised in rabbits against residues 1–14 of the human/mouse CB1 receptor (1:500; kindly given by A. Howlett, North Carolina Central University, Durham, North Carolina, USA) or residues 20–33 of the human/mouse CB2 receptor (1:2,000; Cayman Chemical, Ann Arbor, Michigan, USA) as described (21). Antigen preabsorption experiments were performed by preincubating (37°C, 1 hour) 1 μl (= 0.5 μg) of the anti-CB1 or anti-CB2 Ab and 100 μl PBS with or without 20 μl (= 20 μg) of the corresponding immune peptide (Cayman Chemical). Western blots were subsequently done with the aforementioned Ab dilutions. To determine EGF-R phosphorylation the blots were incubated with anti-phosphotyrosine (1:500; 4G10 mAb; Upstate Biotechnology Inc., Lake Placid, New York, USA) or anti-keratin 5 (1:1,000; Berkeley Antibody Co., Richmond, California, USA) Ab’s, the latter used as a loading control. In all cases, samples were subjected to luminography with an enhanced chemiluminescence detection kit (Amersham Life Sciences, Arlington Heights, Illinois, USA). Densitometric analysis of the blots was performed with the Molecular Analyst software package (Bio-Rad Laboratories Inc., Hercules, California, USA).
Immunohistochemistry. Tissues were fixed in 10% buffered formalin and embedded in paraffin. Sections of mouse and human skin and tumors were stained with the aforementioned Ab’s against CB1 (1:300) and CB2 (1:300) receptors. Control immunostainings using the secondary Ab in the absence of the primary Ab were routinely performed. In addition, antigen preabsorption experiments were carried out with the corresponding blocking peptides as described above. Immunodetection of blood vessels in cryosections of mouse tumors was performed with an anti-CD31 Ab (1:40; PharMingen, San Diego, California, USA). Sections were incubated with a biotinylated anti-rabbit (CB1 and CB2) or anti-rat Ab (CD31) and then with peroxidase-conjugated streptavidin (LSAB Kit Peroxidase; DAKO A/S, Glostrup, Denmark). Ab localization was determined using 3,3′-diaminobenzidine (Vector Laboratories, Burlingame, California, USA). Morphometric values were obtained by examination of six 0.11-mm2 sections per tumor with the image analysis system Leica Qwin (Leica Microsystems Inc., Chantilly, Virginia, USA).
Northern blot analysis. Total RNA was extracted from the tumor samples by the acid guanidinium method (27). The VEGF probe has been described previously (7). The placental growth factor (PIGF) probe was kindly given by G. Persico (Istituto Internazionale di Genetica e Biofisica, Naples, Italy). Probes for angiopoietin 1 (Ang1) and angiopoietin 2 (Ang2) detection were kindly provided by G.D. Yancopoulos (Regeneron Pharmaceuticals, Tarrytown, New York, USA). A 1-kb fragment of the 5′ region of the hEGF-R cDNA was used as probe for EGF-R detection (9). Ribosomal 7S RNA was used as a loading control. Densitometric analysis of the blots was performed with a PhosphorImager using Quantity One software (Bio-Rad Laboratories Inc.).
Statistics. Results shown represent means ± SD. Statistical analysis was performed by ANOVA with a post hoc analysis by the Student-Neuman-Keuls test. Data in Table 1, Figure 5a, and Figure 6c were analyzed by the Mann-Whitney (Wilcoxon) W test to compare medians for nonparametric data.
Figure 5Cannabinoids inhibit skin tumor growth in vivo. PDV.C57 cells were injected subcutaneously in mice. When tumors had reached the desired size, animals were treated with either vehicle (Co), WIN-55,212-2 (WIN), or JWH-133 (JWH), for 11 days. (a) Tumor size (n = 8 for each experimental group). *Significantly different (P < 0.01) from control mice. (b) Examples of subcutaneous tumors in the flank of mice after the indicated treatments. (c) Appearance of tumors dissected from mice after the indicated treatments.
Figure 6Cannabinoids inhibit angiogenesis in skin tumors in vivo. PDV.C57 cells were injected subcutaneously in mice. When tumors had reached the desired size, animals were treated with either vehicle (Co), WIN-55,212-2 (WIN,) or JWH-133 (JWH) for 11 days. (a) Northern blot of the proangiogenic factors VEGF, PIGF, and Ang2. C1, C2; J1, J2; W1, W2 designate tumors from two different animals of each experimental group, that is, treated with vehicle (Co), JWH-133, or WIN-55,212-2, respectively. OD values relative to those of loading controls are given in arbitrary units. 7S, Ribosomal 7S RNA. (b) CD31 immunostaining. Note that control carcinomas show dilated blood vessels, while vessels of cannabinoid-treated tumors are narrow. (c) Morphometric analysis of tumor vasculature (n = 4–6 for each experimental group). *Significantly different (P < 0.05) from control mice.
Table 1Quantification of apoptotic and proliferative cells in vehicle- and cannabinoid-treated skin carcinomas
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Results
Cannabinoid receptors are expressed in skin and skin tumors. The expression of cannabinoid receptors in epidermal cell lines, normal skin, and skin tumors of mice and humans was examined by Western blot analysis and immunohistochemistry. Western blot experiments showed that CB1 and CB2 receptors were expressed in a number of tumorigenic and nontransformed epidermal cell lines of murine and human origin (Figure 1a). In addition, the two receptors were present in normal mouse skin as well as in benign (papillomas) and malign (squamous cell carcinomas) mouse skin tumors. Likewise, CB1 and CB2 receptors were expressed in human skin, keratinocytes, and carcinomas (Figure 1a). To ascertain the specificity of the cannabinoid receptor Ab’s used in the blotting experiments, antigen preabsorption experiments were carried out with the corresponding blocking peptides. As shown in Figure 1b, the peptides blocked anti-CB1 and anti-CB2 Ab binding, not only in skin-derived samples but also in other cell types used as well-established controls for the presence of CB1 (rat cortical neurons) (18), CB2 (human promyelocytic HL60 cells) (19), and CB1 plus CB2 (rat C6 glioma cells) (21).
Immunocytochemical analyses showed that in mouse (Figure 2a) and human (Figure 3a) normal skin CB1 and CB2 receptors were mostly present in suprabasal layers of the epidermis and hair follicles. Basal staining was also observed in some sporadic regions. CB1 and CB2 receptor immunoreactivity was also evident in both papillomas and squamous cell carcinomas of mouse origin (Figure 2a), as well as in human basal cell carcinomas and squamous cell carcinomas (Figure 3a). The specificity of the immunolabeling was shown by experiments in which the primary Ab was omitted and—as mentioned above for Western blots—by antigen preabsorption experiments carried out with the corresponding blocking peptides (Figures 2b and 3b).
Figure 2Immunohistochemical analysis of cannabinoid receptor expression in mouse normal skin and skin tumors. (a) Immunolocalization of CB1 and CB2 receptors. (b) Controls without primary Ab (only with secondary biotinylated anti-rabbit Ab), as well as controls with the Ab-blocking peptides, are shown (see Methods and Results for explanation). Normal skin came from a 5-day-old mouse. Papillomas were generated by chemical carcinogenesis (as described in ref. 9), while SCCs were generated by inoculation of PDV.C57 epidermal tumor cells as described in Methods. Images of representative samples are shown. Similar results were obtained in at least two other samples. Hf, hair follicle; bl, basal layer; sbl, suprabasal layers.
Cannabinoid receptor activation induces skin tumor cell apoptosis. We tested the functionality of cannabinoid receptors in the induction of apoptosis in skin tumor cells. The mixed CB1/CB2 agonist WIN-55,212-2 decreased the viability of the tumorigenic epidermal cell lines PDV.C57 and HaCa4 (Figure 4a). Values of cell viability after WIN-55,212-2 treatment (as percentage of the corresponding incubations with no additions) were 74 ± 10 (day 3) and 62 ± 7 (day 4) for PDV.C57 cells, and 75 ± 7 (day 3) and 71 ± 4 (day 4) for HaCa4 cells. Of interest, the cannabinoid was unable to induce any statistically significant change in the viability of MCA3D and HaCat cells, two nontransformed epidermal cell lines, and of primary human keratinocytes (Figure 4b). The mixed CB1/CB2 agonist HU-210 and the selective CB2 agonist JWH-133 (Ki = 677 nM for CB1 and 3.4 nM for CB2; ref, 25) (both at 25 nM) also induced PDV.C57 death to an extent similar to that of WIN-55,212-2 (data not shown). WIN-55,212-2–induced death of PDV.C57 cells occurred by a process of apoptosis, as determined by oligonucleosomal DNA fragmentation (Figure 4c) and TUNEL staining (Figure 4d).
Figure 4Cannabinoid receptor activation induces skin tumor cell apoptosis. (a) The tumorigenic epidermal cell lines PDV.C57 (open symbols) and HaCa4 (closed symbols) were cultured with 25 nM WIN-55,212-2 alone (circles), or in combination with 0.2 μM SR141716 (SR1) (squares), or 0.2 μM SR144528 (SR2) (triangles), and cell viability was determined (n = 5). (b) The nontransformed epidermal cell lines MCA3D (open circles) and HaCat (closed circles), as well as primary human keratinocytes (closed squares), were cultured with 25 nM WIN-55,212-2, and cell viability was determined (n = 4). (c and d) PDV.C57 cells were cultured as described before, and oligonucleosomal DNA fragmentation (c) (n = 6), and TUNEL staining (d) (one representative experiment of four) were assessed. *Significantly different (P < 0.01) from control incubations.
To evaluate the possible implication of the CB1 and CB2 receptors in cannabinoid-induced apoptosis, the effect of selective receptor antagonists was studied. Thus, the CB1 antagonist SR141716 and the CB2 antagonist SR144528 prevented WIN-55,212-2–induced apoptosis of PDV.C57 cells (Figures 4, a, c, and d), pointing to the involvement of both receptors in the apoptotic action of cannabinoids.
Cannabinoids inhibit skin tumor growth in vivo. Given the inhibition of tumorigenic epidermal cell survival in culture by cannabinoids, we evaluated the effect of cannabinoid treatment on skin tumor growth in vivo. Tumors generated by inoculation of the highly malignant PDV.C57 cell line were treated with vehicle or WIN-55,212-2. As shown in Figure 5, a–c, cannabinoid administration blocked the growth of tumor cells in vivo (in ≈75% of the mice treated).
Because cannabinoid-based therapeutic strategies should be as devoid as possible of psychotropic side effects and PDV.C57 express functional CB2 receptors, we administered to mice the selective CB2 agonist JWH-133. Previously, we have provided pharmacological, biochemical, and behavioral evidence that JWH-133 activates selectively the CB2 receptor and does not elicit psychotropic effects in mice (22). As shown in Figure 5, a–c, tumors from JWH-133–treated animals were significantly smaller than those from vehicle-treated controls (in ≈70% of the mice treated).
We next examined whether, as occurs in cultured skin tumor cell lines, cannabinoids induce apoptosis of malignant cells in vivo. As shown in Table 1, quantification of apoptotic cells in tumor sections revealed that treatment with WIN-55,212-2 or JWH-133 increased the number of apoptotic cells. In contrast, the proliferation index did not significantly differ between control and cannabinoid-treated carcinomas.
Cannabinoids inhibit skin tumor angiogenesis in vivo. Tumors require an adequate supply of oxygen and nutrients to grow more than a few millimeters. For that purpose they produce proangiogenic factors that promote the formation of new blood vessels (28, 29). We therefore analyzed whether the vascularization of growth-arrested cannabinoid-treated tumors was affected. Northern blot analyses showed that the expression of major proangiogenic factors, namely VEGF, PIGF, and Ang2, was strongly depressed by treatment with WIN-55,212-2 or JWH-133 (Figure 6a). The mRNA expression of Ang1 and the two antiangiogenic factors, thrombospondin 1 and thrombospondin 2, was not significantly affected by cannabinoid administration (data not shown). Furthermore, although immunostaining of CD31, a marker of endothelial cells, revealed no significant differences in vascular density (number of blood vessels per unit area) between control and WIN-55,212-2– or JWH-133–treated tumors (Figures 6, b and c), important differences were observed when vessel morphology was examined: while control carcinomas showed a network of dilated vessels, cannabinoid-treated tumors displayed a pattern of blood vessels characterized predominantly by narrow capillaries (Figure 6b). Morphometric analyses confirmed that cannabinoid treatment induced a statistically significant decrease in blood vessel size, as determined by the total area occupied by vessels, the area per vessel, and the vessel larger diameter length (Figure 6c).
Cannabinoids decrease EGF-R activation in skin tumors in vivo. We have recently found that in skin carcinomas EGF-R plays an important role in triggering the angiogenic switch necessary for skin tumor growth (9). Thus, we measured the expression levels and activation state of EGF-R in control and cannabinoid-treated skin tumors. While EGF-R mRNA was highly expressed in vehicle-treated tumors, in line with its known overexpression in skin carcinomas (30, 31), the levels of EGF-R mRNA in cannabinoid-treated tumors were very low (Figure 7a). In addition, the degree of EGF-R activation (autophosphorylation) was markedly reduced in cannabinoid-treated tumors (Figure 7b). Moreover, exposure of cultured PDV.C57 cells to WIN-55,212-2 or JWH-133 blunted EGF-R phosphorylation (Figure 7c), supporting the direct impact of cannabinoids on skin tumor cells.
Figure 7Cannabinoids inhibit EGF-R activation in skin tumors in vivo. (a and b) PDV.C57 cells were injected subcutaneously in mice. When tumors had reached the desired size, animals were treated with either vehicle (Co), WIN-55,212-2, or JWH-133 for 11 days. Northern (a) and Western blot (b) analyses show that EGF-R mRNA expression and EGF-R activation (autophosphorylation), respectively, are severely diminished in cannabinoid-treated tumors. One representative experiment of three is shown in each panel. (c) PDV.C57 cells were cultured for 24 hours with either vehicle, 25 nM WIN-55,212-2, or 25 nM JWH-133, and EGF-R phosphorylation was determined by Western blot analysis. One representative experiment of three is shown. OD values relative to those of loading controls are given in arbitrary units. Ribosomal 7S RNA (7S) and keratin 5 (K5) were used as loading controls in Northern and Western blots, respectively. PY, phosphotyrosine.
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