Morphology and Immunofluorescence Staining of Human Cultured Melanocytes
In all protocols, the cultured cells of passages 1-6 had bi- or tri-polar multidendritic morphologies, in contrast to melanoma cell lines which had polygonal and epithelioid-like morphologies (Figure 1A). Immunofluorescence staining confirmed the expressions of Melan-A, S100 protein, HMB-45, and tyrosinase II in the cytoplasm of passages1, 3 and 6 melanocytes cultured using each of the four protocols. The expressions of Melan-A, S100 protein, HMB-45, and tyrosinase II were confirmed by Image J software in > 90% of cultured melanocytes. Furthermore, A375, NA8, and D10 melanoma cell lines expressed Melan-A and S100 surface markers, however, the cells were negative for HMB-45 and tyrosinase II markers. All the fibroblasts lacked melanocyte surface markers."(Figure 1B).
PDT Assessments showed that protocols A and B are the most appropriate
The mean PDT of passage-1melanocytes cultured using protocols A, B, C, and D were 34.8, 33.0, 24.7, and 22.9 hours, respectively. As pointed out in Figure 2, these values were persistent from passage 1 to passage 6, while at passage 6, PDT for protocols C and D were 23.6 and 22.0 hours which were significantly lower than those of protocols A and B (39.1 and 36.2 hours, respectively) (P<0.001). As shown in (Figure 2 A) proliferation of cultured melanocytes from primary culture to passage-3 following utilization of all protocols, showed non-significant consistent growth ranging from 10,000 to 20,000 cells/ml. However, cells cultured using protocols C and D exhibited a sudden increase in number especially in passages-4 5 (i.e. 40,000 to 60,000 cells/ml, respectively) in contrast to that achieved following utilization of protocols A and B, in which the number of cells remained unchanged till passage 6. (Figure 2 B)
Karyotype Analysis
Karyotypes of passages-5 and 6 melanocytes obtained from 8 patients displayed no structural alterations in the 15 metaphase plate spreads All chromosomes were normal and none showed instability. In contrast, the positive control melanoma cell lines NA8, A375, and D10 showed numerous structural alterations that included deletions, duplications, and rearrangements, as well as aneuploidy and polyploidy. The NA8 melanoma cell line displayed a modal distribution of chromosome numbers 59-61 (hypotriploid), A375 cells displayed a modal distribution of chromosome number 62 (hypotriploid), and D10 cells displayed a bimodal distribution of chromosomes 46–48 (near triploid, Figure 3A and Table 4).
Table 4
Karyotype results detected in the melanoma cell lines.
Cell line
|
Karyotype
|
Chromosomal instability signs
|
NA8
|
Modal no: 59-61 (Hypotriploid)
|
59-61,XX,-2[4],t(2;13)(p11.2;q11)[4],-3[4],t(3;17)(p25;q25.2)[4],t(3;17)(p27;q25.2)[4], +4[4],del(4)(q33)[2],del(4)(q33)[1],dup(4)(q21q24)[4],+5[4],del(5)(p15)[4],rev(5)(p?)[4],-6[4],del(7)(q31)[4],der(7)t(7;10)(q32;q23)[4],8[4],+10[4],del(10)(p13)[4],del(10)(p13)[4],add(11)(p11.2)[4],der(12)t(1;17)(q21;p11.1)[4],der(12)t(9;12)(q21;p13)[2],add(13)(p13)[3],der(13)t(5;13)(q11.2;p13)[2],-14[3], add(14)(q32)[2],t(14;14)(q10;q10)[1], -15[3],add(15)(q13)[14],der(15)t(15;21)(p13;q11.2)[4],-16[2],-18[2],add(18)(q23)[4],-19 [2],-21,-22[2],+mar, mar.
|
A375
|
Modal no: 62 (Hypotriploid)
|
62,XX,+1[3],+del(2)(q32)[4],+3[4],+5[4],der(6)t(1:6)(q12:q13)[4],+7[1],+8[4],+9[3], +10[2],inv(11)(q12q22)[2],+12[4],+13[3],+14[4],+15[4],+16[4], add(16)(q24) [1],+17[3],+18[3],add(19)(q13.4)[4],+20[4],-22[3],+mar[1],+X[1][cp4].
|
D10
|
Modal No: 46-48 (Near triploid)
|
46-48,XX,i(1)(q10)[1],+der(2) t(2:X)(q21;q3) [3],dup(5)(q32;q34)[4],der(7)(q32)[4],-9[4],del(10)(p12;3)[4],t(12;15)(q10;q10),add(13)(p13)[4],der(13) t(1;13)(p13;q12)[4],der(14) t(1;14)(p11.2;p13)[4],+5[4],del(16)(p12)[4],-17[1],+20[1],-X[4][CP4].
|
Gene Expression Analysis
BRAF, NRAS, and HRAS gene expressions in A375 and D10 melanoma cell lines and cultured melanocytes were assessed by RT-PCR. We observed <1-fold increases in HRAS gene in passage 3 of cultured melanocytes. Also, in passages 1 and 6, expression levels of HRAS gene showed less than 2-fold increases. Moreover, BRAF gene in cultured melanocyte of passages 1, 3, and 6 showed 2-fold increases. Interestingly, passage 3 showed the lowest level of BRAF gene expression. In comparison, BRAF gene expression of melanoma cell lines A375 and D10 showed 8 fold increase which is of crucial importance in melanomas (p<0.05). Also, the data showed 1-fold increase in the expression of NRAS gene of passages 1 and 3 of cultured melanocytes Overall, the NRAS gene expression was significantly increased (up to 5 fold) in passage 6, in melanoma cell lines A375 and D10, NRAS gene expression showed a 6-fold increase. (Figure 3B)
Gene Sequencing
We investigated probable mutations in BRAF exon 15 and NRAS exons 1 and 2 in cultured melanocytes (passages 5 and 6) Sequencing analysis showed that in melanoma cell lines A375 and D10, BRAF gene had a T to A transversion at nucleotide position (BRAF c.1799T>A) or amino acid position 600 which resulted in an amino acid substitution from Valine to Glutamic Acid at codon 600 (V600E). None of the cultured melanocyte chromatograms had mutations at V600E (Figures 4 A-C). DNA sequence of the NRAS gene in exons 1 and 2 did not show any mutations neither in the cultured melanocytes nor the A375 and D10 melanoma cell lines (Figures 5 A-C). Genome sequencing of cultured melanocyte showed that all nucleotides were in correct positions and no mutations were seen. All passage-6 melanocytes obtained from eight patients were analyzed and no wrong nucleotide was seen.
Presence and Pigmentation of Cultured Melanocytes in Albino Mice
(Figure 6A) shows the punctuated pigmentation on injected site of the skin, which indicates the functionality of cultured cells after transplantation. The histological assessment showed intra-epidermal existence of the transplanted cells. The immunohistochemistry results showed bioactive cells after transplantation. The results showed that 96% (19 of 20) of cells were positive for Melan-A, 92% (18 of 20) of cells were positive for S-100, and 81% (15 of 20) of cells were positive for HMB45 in 20 melanocytes counted. The Immunostaining results confirmed the presence of cells in epidermis. (Figure 6B)
Tumorigenicity Assessment of Cultured Melanocytes in BALB/c Nude Mice
Cultured melanocytes of passages 5 and 6 were injected into BALB/c nude mice. Also, melanoma cell lines namely, A375, D10 and NA8 and fibroblasts were injected as positive and negative controls, respectively. We monitored the mice weekly over a period of 16 weeks, it was observed that mice injected either with cultured melanocytes (cultured melanocytes/animal, n= 48) or fibroblasts had no visible evidence of tumor formation, whereas palpable tumors were readily detected at the injection site on the scapula in mice that received A375, D10 and NA8 cells (Figure 7A).
Size and appearance of tumors were similar to those of melanoma tumors, however, following transplantation of cultured melanocyte into nude mice, no tumors, nodules or any palpable tissue were seen. This monitoring was continued for four months. To make sure, we investigated possible BRAF mutation at codon 600 (V600E) after 16 weeks of transplantation in biopsy samples obtained from mice with melanoma and cultured melanocytes groups. The BRAF V600E mutation that resulted in an amino acid substitution from valine to glutamic acid, was found in all of the samples from the melanoma groups. However, gene sequencing of the BRAF gene in nude mice that received the melanocytes showed normal results. And the result of Histopathological tissues analyses suggested that following subcutaneous injection, the integration of the cultured melanocytes of passages 5 and 6 into the subcutaneous tissue occurred without non-transformed cells and no pigmentation. In contrast transplantation of human melanoma cell lines showed cells expressed antibodies. (Figures 7 B-D).