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Departments of 1 Immunology, 2 Pathology, and 3 Preventive Medicine, Nagoya University Graduate School of Medicine, Nagoya; 4 Department of Tissue and Organ Development, Regeneration and Advanced Medical Science, Gifu University Graduate School of Medicine, Gifu; and 5 Department of Environmental and Preventive Medicine, Graduate School of Medical Science, Kanazawa University, Kanazawa, Japan
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ABSTRACT |
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 Top ABSTRACT IntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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We have shown that receptor tyrosine kinase Ret/RET is physiologically expressed in melanocytes in the inner and outer sheaths of hair follicles and matrix of hair bulbs of the skin of normal C57BL/6 mice and humans in close association with both melanin production and hair growth of the anagen phase (6) . These findings suggest that Ret/RET kinase is involved in the physiological mechanism of melanocyte activation and melanin production. On the other hand, it has been established that another protein tyrosine kinase, c-Kit/c-KIT, plays a crucial role in skin melanogenesis for both melanocyte entry into the epidermal layer at a restricted prenatal stage and for melanocyte activation during postnatal life (7, 8, 9, 10, 11) . It has been reported that c-KIT is expressed in human normal melanocytes, benign nevi, some dysplastic nevi, and early stage of melanoma (12 , 13) . However, c-KIT expression is lost in the late stage of melanoma (12, 13, 14) . This fact argues against the view that c-Kit plays a crucial role in the process of development of melanoma. Results of our earlier results suggested that c-Kit and RET collaborate in melanocyte activation and development of benign melanocytic tumors (15) . However, the potential role of c-Kit/c-KIT in the development of melanoma is not known. Using a recently established new subline (line 304/B6) of RET-transgenic mice in which melanoma develops after development of benign melanocytic tumors (5) , we found that, not closely linked to the known function for melanocyte activation, c-Kit plays a crucial role in the oncogene RET-dependent development of melanoma, and we describe here a novel immunotherapy-targeting c-Kit at the neonatal stage that strongly suppresses melanoma development.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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Western Blotting.
Western blotting of c-Kit and RET was performed with skin lysates according to the method described previously (18)
. The lysates (30 µg/lane) were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes (Nihon Millipore KK, Yonezawa, Japan). After the membranes had been reacted with anti-c-Kit or anti-Ret (Immuno-Biological Laboratories, Gunma, Japan) antibody, the reaction was examined using Western Blot Chemiluminescence Reagent (DuPont NEN, Boston, MA).
Histology and Immunohistochemistry.
Skin sections were stained with H&E, and immunohistochemistry was performed with anti-c-Kit or -S100 protein antibody as described previously (17)
.
Statistical Analysis.
Difference of tumor volumes in original RET (304/B6)-transgenic mice, Wv/+-RET (304/B6)-transgenic mice, and Wv/Wv-RET (304/B6)-transgenic mice (Fig. 1D)
or ACK2-treated and untreated RET (304/B6)-transgenic mice (Fig. 3A)
in each month was statistically analyzed by the Mann-Whitney U test, as shown in the previous study (19)
. Tumor volumes in mice that died before the statistical comparison in each month were calculated as a continuation of the volume up to the month of comparison (Figs. 1D
and 3A)
. Difference of life spans in original RET (304/B6)-transgenic, Wv/+-RET (304/B6)-transgenic, and Wv/Wv-RET (304/B6)-transgenic mice (Fig. 1F)
was statistically analyzed by Log-rank test. Difference of benign and malignant tumors incidence within 18 months after birth in original RET (304/B6)-transgenic, Wv/+-RET (304/B6)-transgenic, and Wv/Wv-RET (304/B6)-transgenic mice (Fig. 1G)
or in ACK2-treated and untreated RET (304/B6)-transgenic mice (Fig. 3C)
was statistically analyzed by the 
2 test.
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Results and Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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, were characterized by anemia, sterilization, and disturbance of mast cell, B-cell, and melanocyte development (7, 8, 9, 10, 11
, 20
, 21)
. There was basically no difference in the characteristics regarding sterilization and disturbance of B-cell development in bone marrow between the Wv/Wv mice and newly produced Wv/Wv-RET (304/B6)-transgenic mice in our preliminary results.6
However, the Wv/Wv-RET (304/B6)-transgenic mice had a mosaic of white and black hairs/skin (the area of black hairs/skin occupying 5–70% of the total skin area of an individual mouse; Fig. 1A
) and excess melanogenesis in and around hair bulbs in the area of black hairs/skin (Fig. 1B)
, apparently not less extensively than did original line 304/B6 RET-transgenic mice (6)
. Interestingly, the area of black hairs but not that of white hairs clearly expressed RET protein (Fig. 1, A and C)
. It is known that c-Kit function is required for both melanocyte entry into the epidermal layer during midgestation and for melanocyte activation linked to the hair growth cycle during the postnatal phase (9
, 22)
. The c-Kit Wv protein is indistinguishable from normal c-Kit protein with regard to size and cell surface expression, but its in vitro kinase activity is 
10–20% of that in +/+ cells (8)
. It was thought from the data on hair/skin color of Wv/Wv-RET (304/B6)-transgenic mice that severe impairment of c-kit function partially blocked the entry of melanocytes into the epidermis in the presence of activated RET but did not greatly reduce the RET-mediated excess melanin production in melanocytes that entered epidermis. We compared the long-term (30 months) profiles of the time-dependent development of melanoma in line 304/B6 RET- and Wv/Wv-RET (304/B6)-transgenic mice (Fig. 1, D and E)
. Unexpectedly, from the rather small effect of severe impairment of c-Kit-function on RET-mediated excess melanin production, even benign melanocytic tumors failed to develop in 38 (86.3%) of the 44 Wv/Wv-RET (304/B6)-transgenic mice throughout their lifetime (Fig. 1G)
. At 12 months after birth, none of the Wv/Wv-RET-transgenic mouse had died of melanoma growth, whereas 28 (70%) of the 40 parental line 304/B6 RET-transgenic mice had died from rapidly growing melanoma. Melanocytic tumors that had developed in only 8 of the 44 Wv/Wv-RET (304/B6)-transgenic mice grew very slowly except in 2 mice, which died of tumor growth at a late time (at 16.5 and 23.1 months old). The tumor-free period of the Wv/Wv-RET (304/B6)-transgenic mice (mean ± SD: 13.8 ± 4.6 months) was much longer (P < 0.0001) than that of line 304/B6 RET-transgenic mice (3.7 ± 1.3 months). The life span of the former (15.8 ± 5.9 months; n = 25) was significantly longer (P < 0.0001; n = 50) than that of the latter (9.7 ± 3 months; n = 50; Fig. 1F
). Even the Wv/+-RET (304/B6)-transgenic mice (13.2 ± 4.8 months; n = 50) had a significantly (P < 0.0001) prolonged life span, compared with that of original RET (304/B6)-transgenic mice (Fig. 1F)
. The mean life span of the Wv/Wv-RET (304/B6)-transgenic mice was not significantly shorter than the mean life span of parental Wv/Wv mice (15.7 ± 4.9 months; n = 10), which was much shorter than that of conventional C57BL/6 mice (>24 months), probably because of impairment of c-Kit-dependent hematopoietic stem cell activity (7)
. These results demonstrated that severe impairment of c-Kit function rescued the majority (42 of 44 mice) of RET (304/B6)-transgenic mice from otherwise destined death caused by hereditary melanoma (P < 0.0001).
Prolongation of the tumor-free period (11.9 ± 4.9 months) and life span (13.2 ± 4.8 months) to lesser degrees and reduction of overall tumor growth to a lesser degree (Fig. 1D)
were also observed in Wv/+ RET (304/B6)-transgenic mice (n = 61), in which c-Kit function was thought to be impaired in >50% of the mice. Melanin was produced in skin/hair of these mice almost as extensively as that in skin/hairs of original line 304/B6 RET-transgenic mice (data not shown). Nevertheless, no detectable tumors developed in 19 (31.1%) of the 61 Wv/+ RET (304/B6)-transgenic mice throughout their lifetime, and only 6 (9.8%) and 25 (40.9%) of the mice died of growing melanoma within 12 and 18 months after birth, respectively. c-Kit ligand function-impaired Sld/Sld- and Sld/+-RET (304/B6)-transgenic mice also displayed basically the same phenotype as did Wv/Wv- and Wv/+-RET (304/B6)-transgenic mice (data not shown). These results indicated that c-Kit function is essential for oncogene RET-dependent melanoma development and that this c-Kit function, unlike the c-Kit function needed for melanin production, is not able to be compensated by RET. It was noted that life-threatening melanoma barely or rarely developed in Wv/Wv- and Wv/+RET (304/B6)-transgenic mice even in areas with excess melanin production, in which RET was clearly expressed (Fig. 1C)
and compensated the impaired c-Kit function for excess melanin production by melanocytes localized in skin (Fig. 1B)
. These results argued against the view that c-Kit function is required for melanoma development simply through its actions to localize RET-expressing melanocytes in the skin and activate them for melanin production. It seems that the threshold of the requirement of c-Kit for melanoma development is higher than that for skin localization and melanin production by melanocytes, possibly because some c-Kit function other than that needed for the physiological dynamics of melanocytes is required for the signaling to melanocytes for oncogenesis.
The true reason why melanoma would not develop in the Wv/Wv-RET (304/B6)-transgenic mice is still unknown. Host-tumor interactions often play an important role in tumor control and elimination. In fact, the establishment of tumor immunity for RET oncogene product is crucially involved in the mechanism to suppress the tumor development in line 242 of RET-transgenic mice (17 , 23) . The Wv/Wv-RET (304/B6)-transgenic mice, in which c-Kit protein with a mutation expresses, may cause the mutant c-Kit-specific immune responses, which lead to the suppression of development. Anyhow, further study is required to clarify the mechanism of c-Kit mutation-linked suppression of melanoma development in the Wv/Wv-RET (304/B6)-transgenic mice.
c-Kit Expression Is Promoted by RET Soon after Birth.
We showed previously that both innate c-Ret and RET in RET-transgenic mice are transiently expressed at high levels soon after birth in association with primary hair growth (6
, 17)
. c-Kit was also reported to be expressed in melanocytes in skin soon after birth (22)
. We compared the levels of c-Kit expression in skin of RET-transgenic and littermate nontransgenic mice soon after birth. As shown in Fig. 2, A and B
, c-Kit expression levels in RET-transgenic mice, which were determined by either immunohistochemitry (A) or Western blotting (B), were elevated compared with those in nontransgenic mice, suggesting that RET promoted expression of c-Kit in the skin. Together with our results that impairing the c-Kit function would inhibit tumor growth in the RET (304/B6)-transgenic mice (Fig. 1D)
, these results suggest that c-Kit works in the downstream of RET-mediated signal transduction for the development of melanoma. As shown in Fig. 2C
, benign melanocytic tumors expressed high levels of RET, which decreased when the tumors became melanoma, in agreement with our findings reported previously (23)
. Benign melanocytic tumors, in which RET was expressed at high levels, expressed as a high level of c-Kit, as did the skin obtained from RET-transgenic mice soon after birth, which decreased in melanoma. These results suggest that protein expression levels of c-Kit and RET bidirectionally control melanoma development. The down-regulation of c-Kit protein expression level at the late stage of melanoma development may permit melanoma cells to avoid stem cell factor/c-Kit-mediated apoptosis, hence contributing to tumor growth. To further evaluate the potentially bidirectional roles of c-Kit during the melanoma development and study the relationship between the roles of RET gene and c-Kit gene, establishment of tumor cell lines derived from RET (304/B6)-transgenic mice and Wv/Wv-RET (304/B6)-transgenic mice would be useful.
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, although tumors developed in some mice at a later stage (Fig. 3A)
. In 13 of the 28 ACK2-treated mice, even benign melanocytic tumors did not develop within 18 months after birth (Fig. 3C)
. The tumor-free period of the treated mice (11.1 ± 1.7 months) was much longer (P < 0.0001) than that of the untreated control mice (3.7 ± 1.3 months). At 12 months after birth, >70% of the control RET-transgenic mice (n = 46) had died of tumors, but none of the 28 ACK2-treated transgenic mice had died from rapidly growing melanoma. At 18 months after birth, only 9 (32.1%) of the 28 ACK2-treated mice had died from melanoma (Fig. 3C)
. The life span of the treated mice (14.2 ± 3.2 months) was 46% longer (P < 0.0001) than that of the control mice. Injection of ACK2 at an early time after birth seems to be critical because the number of deaths from tumors in RET-transgenic mice that had been given an injection of ACK2 within 24 h after birth (4 of 18, 22.2%) was fewer than that in mice that had been given an injection 3 days after birth (4 of 9, 44.4%).
Corresponding to the unexpectedly long lasting effect of ACK2 injection for melanoma suppression, the treatment caused an unusually long lasing change in hair color of the transgenic mice. As shown in Fig. 4
, hair color of nontransgenic control mice (No. 1 and No. 2 mice in Fig. 4, A–D
) had changed from black (No. 1) to gray/white (No. 2) at 4 days after ACK2 injection (Fig. 4B)
, and the change was most evident at 12 days after ACK2 injection (Fig. 4C)
. This change in color, which agrees with a finding reported previously (22)
, was reversed almost to the native color (black) within a few months (Fig. 4D)
. Interestingly, the hair color of RET-transgenic mice (No. 3 and No. 4 mice) had changed from deep black (No. 3) to gray (No. 4) at 4 days after ACK2 treatment (Fig. 4B)
and at 12 days after the treatment (Fig. 4C)
and, more importantly, had not returned to black at 3 months after (Fig. 4D)
or even at 8 months (Fig. 3B
, top) to 18 months (data not shown) after the injection. Correspondingly, the number of S100-containing melanocytes and amount of melanin pigment in the skin of 12-month-old RET-transgenic mice (Fig. 4, G and I)
, which were much larger than those in littermate nontransgenic mice (Fig. 4E)
, were reduced by injection of ACK2 soon after birth, and these changes were not reversed at 12 months after the injection (Fig. 4, H and J)
. In contrast, the effect of the injection of ACK2 almost disappeared in nontransgenic control mice in a few months (Fig. 4, E and F)
. These observations demonstrated that overall c-Kit function was decreased more by the ACK2 treatment than by Wv mutation, resulting in reduction of not only melanoma development but also melanocyte activation in the presence of RET and that this change was maintained for an unusually long time in ACK2-treated RET-transgenic mice.
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. These results might encourage us to use a c-KIT-specific antibody in the future to treat some cases of human hereditary melanoma, such as congenital melanocytic nevus in which c-KIT expression in melanocyte lineage cells and their precursors may be promoted in a synchronized manner by RET-equivalent oncogenes. c-KIT could also be a target molecule for prevention and treatment of melanoma in general, although a preliminary trial of application of ACK2 injection to already established melanoma-bearing RET-transgenic mice was not successful.7 In addition, the results of our study suggest a new principle for use of monoclonal antibodies in treating congenital diseases through eliminating "sick" cells that may express targeting antigens for immunotherapy in a synchronized manner.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Masashi Kato, Department of Environmental and Preventive Medicine, Graduate School of Medical Science, Kanazawa University, 13-1 Takaramachi, Kanazawa, Ishikawa 920-8640, Japan. Phone: 81-76-265-2216; Fax: 81-76-234-4233; E-mail: katomasa{at}pub.m.kanazawa-u.ac.jp
6 M. Kato et al., unpublished observations. 
7 M. Kato et al., unpublished data.
Received 9/ 8/03. Revised 11/26/03. Accepted 12/16/03.
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; n = 40), Wv/+-RET (304/B6)-transgenic mice (
; n = 61), and control original line 304/B6 RET-transgenic mice (
; n = 44). *, 
, 
, significantly different (*, P < 0.0001; 
; n = 42). *, 
