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Department of Dermatology, Boston University School of Medicine, Boston, Massachusetts, USA
2Correspondence: Department of Dermatology, Boston University School of Medicine, 609 Albany St., J-Bldg., Boston, MA 02118-2394, USA. E-mail: bgilchre{at}bu.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: melanocyte • apoptosis • differentiation • p73 • livin/ML-IAP
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, a >20-fold increase during the past century. Metastatic melanoma is highly resistant to chemotherapy and is almost always fatal. Molecularly, lesions in the BRAF (2)
and p16INK4A (3)
genes have been linked to the etiology of melanoma, and loss or repression of p16 is common in more advanced stages of the disease (4
, 5)
. The p16 block to proliferation was recently found to be overcome often by amplification of the cdk6 gene in a UV-induced mouse melanoma model (6)
, again showing the importance of the pRb/p16 pathway in melanoma. The p53 tumor suppressor protein is functional in the great majority of early melanomas, but p53 is often mutated in metastatic melanoma and established melanoma cell lines, contributing to chemotherapy resistance of melanoma (7)
. Recent work also implicates overexpression of proteins in the inhibitors of apoptosis (IAP) family in melanoma cells' resistance to chemotherapy and irradiation (8
, 9)
. However, a unifying hypothesis for melanoma progression and treatment resistance is not available.
Mammalian cells are protected from malignant transformation by a variety of protective responses, all of which are customarily triggered by DNA damage. These include a tightly regulated program of replicative senescence that limits the replicative potential of each cell (10)
and a cell suicide program classically activated by extensive and presumptively irreparable DNA damage (11)
. Telomeres have been implicated in both processes.
The 3' end of each telomere consists of a single-stranded overhang (in mammals 
150–200 bases of TTAGGG tandem repeats) that inserts into the proximal double-stranded telomere and stabilizes a loop structure at the chromosome ends (12)
. Disruption of the telomere loop structure by expression of a dominant negative version of the telomere repeat binding factor TRF2, principally responsible for loop integrity, results in degradation of the 3' telomere overhang as well as ATM-mediated induction of the p53 tumor suppressor and transcription factor, leading to responses such as cell senescence and apoptosis, depending on the cell type (13
14
15)
.
In most cancer cells telomere length is maintained by the enzyme telomerase and, in 10%, by a telomerase-independent alternative method. Telomerase is an enzyme complex with at least three components: an RNA template and a telomerase-associated protein that are constitutively expressed and a catalytic subunit, telomerase reverse transcriptase (TERT), which adds TTAGGG residues to the 3' terminus (16
17
18)
. TERT is generally not expressed in normal somatic cells (19)
but is expressed in the great majority of primary tumors and malignant cell lines (20)
. Expression of telomerase confers immortality on cells, but recent work demonstrates that telomerase affects immortalized cells in ways other than maintenance of telomere length that promote their transformation (21)
. Virtually all human melanomas express telomerase (22
23
24
25
26)
; the level of telomerase activity in melanomas often increases as the tumor progresses (24
, 26
, 27)
, culminating in metastases that are highly resistant to chemotherapy.
Cancer therapy is directed at killing malignant cells, often by inducing apoptosis. However, other cellular responses to DNA damage, such as increased differentiation or induction of replicative senescence, may contribute to the therapeutic response. Induced differentiation might be especially helpful in immunotherapy of malignancy, when expression of differentiation antigens such as gp100 and MART-1 by melanoma cells may permit targeting by sensitized T lymphocytes (28
29
30)
.
Previous work from our laboratory has shown that 2- to 11-base DNA oligonucleotides homologous to the telomere 3' overhang, which we term T-oligos, induce DNA damage responses in primary human cells (31
32
33
34
35
36
37
38
39)
. These responses, which are associated with nuclear accumulation of the T-oligos (34
, 36)
, include enhanced DNA repair capacity (32
, 37
, 38)
, reduced UV-induced mutagenesis and photocarcinogenesis in mice (39)
, cell cycle arrest (31
32
33
34)
, a senescent phenotype in fibroblasts (39)
, and increased pigmentation in melanocytic cells (33
, 35
, 36)
. T-oligos can induce apoptosis in immortalized and malignant cell lines (34)
. The T-oligo responses are mediated in large part through activation of the ATM kinase (31)
and p53 (31
, 32
, 34
, 37)
or its homologue p73 (34)
, but do not result in digestion of the 3' telomere overhang (31)
as is observed after experimental telomere loop disruption (14)
. These observations led us to hypothesize that T-oligos mimic the physiologic signal resulting from DNA damage or experimental telomere loop disruption that initiates cancer prevention responses. We further hypothesize that this signal is exposure of the otherwise sequestered telomere 3' overhang.
In this study, we examine the ability of an 11-base T-oligo vs. control complementary or unrelated oligonucleotides or diluent alone to induce apoptosis and differentiation of the aggressive human melanoma cell line MM-AN derived from a metastatic melanoma (40)
. The MM-AN line was selected because it forms subcutaneous tumors in severe combined immunodeficient (SCID) mice (41)
and metastasizes to the lungs, pancreas, brain, and other organs (40)
. Moreover, therapeutic responses in melanoma-bearing SCID mice have been shown to predict clinical responses in human trials (42
, 43)
.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell culture and cell cycle analysis
MM-AN, MU, PM-WK, MM-RU, MM-MC, and RPM-EP melanoma cells were obtained by explant culture (44)
and grown in MEM with 10% FBS (both from Gibco/BRL, Gaithersburg, MD, USA). Normal newborn human melanocytes were established and cultured as controls as described previously (45)
. Duplicate melanoma cell cultures were grown in MEM with 5% FBS and treated for 96 h with a final concentration of 40 µM of oligonucleotides homologous, complementary, unrelated to the telomere 3' overhang sequence or an equal volume of diluent (water) as a control. Cells were collected, stained with propidium iodide, and analyzed by a Becton Dickinson FACS Scan and CellQuest software.
TUNEL analysis
To detect apoptosis by DNA end labeling, cells were grown and treated with the oligonucleotides as described above; 72 h after treatment cells were fixed in 4% paraformaldehyde for 30 min, permeabilized with 0.1% Triton X-100 for 2 min, and the TUNEL assay carried out with a cell death detection kit from Boehringer Mannheim (Indianapolis, IN, USA) using fluorescein-labeled dUTP. TUNEL-positive cells were detected by FACS.
Western blot analysis and antibodies
Melanoma cells were cultured in MEM with 5% FBS and treated with 40 µM of the T-oligo or control (complementary or unrelated) oligo or an equal volume of diluent (water) for various times. Proteins were analyzed by denaturing PAGE (SDS-PAGE) and Western blot analysis as described (33)
using an anti-tyrosinase monoclonal antibody (clone T311, Nova-castra, New Castle upon Tyne, UK), anti-TRP-1 monoclonal antibody (clone Ta99, Signet, Dedham, MA, USA), anti-gp-100 monoclonal antibody (MO 634, Dako Corporation, Carpinteria, CA, USA) and anti-MART-1/Melan-A monoclonal antibody (clone M2-7C10, Signet, Dedham, MA, USA), and anti-livin/ML-IAP goat polyclonal antibody (AB798 Novus Biologicals Inc., Littleton, CO, USA).
Animals
SCID mice were purchased from Fox-Chase Cancer Center (Philadelphia, PA, USA) and hosted in the pathogen-free animal facility at Boston University Medical Center.
Effect of T-oligo on melanoma metastasis
To study the effect of the T-oligo on the metastatic potential of MM-AN cells, they were treated with 40 µM of T-oligo or the complementary sequence or diluent for 48 h in MEM with 5% FBS. The cells were then harvested with trypsin/EDTA and the percent viability was determined by Trypan blue exclusion. Only cell populations with 
90% viability were injected: 1 x 106 cells in balanced salt solution into the lateral tail vein of 5-wk-old SCID mice. There were three groups of five mice. Group 1 was injected with T-oligo-treated cells, group 2 was injected with cells treated with the complementary control oligo, and group 3 was injected with diluent-treated cells. After 40 days mice were killed by CO2 inhalation; an autopsy was performed and organs were examined macroscopically for visible lesions using calipers. Emphasis was given to lung, pancreas, brain, brown fat, kidney, and bone because these organs have been shown to be the primary sites of metastases of MM-AN cells (40)
. For examination and sectioning, organs were rinsed in PBS and fixed in 10% formalin for 48 h. Organs were examined under a dissecting microscope and the number of tumor nodules counted. For histological examination, tumors were fixed in formalin, embedded in paraffin, and sections were stained with hematoxylin and eosin (H&E). Melanocytic origin of the metastases was confirmed by the presence of melanin pigment.
Treatment of SCID mice for studying the effect of T-oligo on tumorigenicity
MM-AN melanoma cells were cultured in MEM with 5% FBS and harvested with trypsin/EDTA. Tumorigenicity of these melanoma cells was determined by intradermally injecting 2 x 106 viable cells in balanced salt solution into the flank region of SCID mice to produce subcutaneous tumors. Three groups of six mice per group were studied. Twice-daily intratumoral injections began 72 h after the melanoma cells were injected: group 1 was given T-oligo (210 µg=60 nmol in 150 µL of PBS), group 2 was given the complementary control oligo; and group 3 was injected with diluent alone. The mice were killed after 22 days, 24 h after the last treatment. The tumors were measured with calipers, fixed in 10% formalin, and stained with H&E. Immunohistochemical staining of tumors was done using gp-100 and TRP-1 monoclonal antibodies. For technical reasons (specific staining only slightly above background), expression of tyrosinase and MART-1 could not be assessed. Apoptosis was determined by TUNEL analysis of tumor cross sections using the ApopTag Kit from Intergen (Purchase, NY, USA). To test the effect of T-oligo administered intraperitoneally (IP), 1.5 x 106 cells were injected subcutaneously into the flank and IP in three groups of eight mice per group. After 3 days, mice were treated twice daily with T-oligo or complementary oligo (420 µg in 100 µL of PBS, equivalent to an initial concentration of 80 µM if injected IV, assuming a 1.5 mL blood volume) or diluent alone. Mice were treated for 25 days, killed, and tumor size was determined.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 34)
. At 72 h the S-phase arrest persisted, but 22% of the MM-AN cells appeared in the sub-G0/G1 region of the FACS profile indicating apoptosis; by 96 h the percent of cells then in apoptosis tripled (Fig. 1
A). Within 72 h, TUNEL analysis demonstrated a distinct increase in fluorescence intensity of cells treated with T-oligo, a second marker of DNA digestion, confirming widespread apoptosis (Fig. 1B
). Neither the control oligos nor diluent alone affected the cells. MM-AN cells do not express detectable levels of p53 (data not shown) but, as reported (34)
, T-oligo-induced apoptosis is preceded by an increase in the E2F1 and p73 transcription factors and is inhibited by 50% in MM-AN cells ectopically expressing a dominant-negative form of p73, suggesting that the apoptosis is mediated at least in part by the interaction of E2F1 and the p53 homologue p73. In contrast, T-oligo did not induce apoptosis in normal human melanocytes within 96 h although it did induce an S-phase arrest, most apparent at 24 h (Fig. 1C
), as reported for another T-oligo, thymidine dinucleotide, in these cells (46)
and for the 11-base T-oligo in other primary cell types and established cell lines (31
, 34)
. To determine whether the MM-AN cell response (64±4% apoptosis at 96 h) was unique, five other melanoma cell lines were examined. Four (MU, PM-WK, MM-RU, and MM-MC) underwent extensive apoptosis (57±2%, 46±3%, 45±4%, 29±2%, respectively) as determined by FACS analysis 96 h after treatment with T-oligo.
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The protein livin, also called ML-IAP, is a member of the potent inhibitor of apoptosis protein family that is commonly up-regulated in melanoma cells and largely responsible for resistance to chemotherapy-induced cell death in at least some cell lines (8
, 9
, 47
, 48)
. MM-AN cells expressed a high constitutive level of livin/ML-IAP; treatment with T-oligo dramatically reduced the expression at 72 and 96 h compared with diluent or control oligos, with small reductions apparent by 24 and 48 h (Fig. 2
). Although Fig. 1
demonstrates DNA degradation within 72–96 h, the reduction in livin/ML-IAP cannot be attributed to extensive nonspecific protein degradation in the cells analyzed, as other proteins in T-oligo-treated cells were selectively up-regulated (see below). Therefore, in this cell type the apoptotic response to T-oligo appears likely to be modulated not only by the induction of the proapoptotic proteins E2F1 and p73, as reported (34)
, but by down-regulation of livin/ML-IAP.
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Progression of melanoma is often associated with the loss of melanocyte-specific antigens, presumptively defeating attempts at immunotherapy (49)
. Because melanocyte differentiation is associated with slower cell proliferation, as observed after treatment with T-oligo (33
, 36
, 46)
, and because 2- to 9-base T-oligos enhance differentiation (melanization) of human melanocytes and S91 murine melanoma cells (33
, 35
, 46)
, we examined the expression of melanocyte differentiation antigens that have been targeted for immunotherapy. Relative to control treatments, by 48–72 h T-oligo induced the melanocyte-associated differentiation markers tyrosinase-related protein 1 (TRP-1 or gp 75) (Fig. 3
A) and glycoprotein 100 (gp 100) (Fig. 3B
), two antigens whose expression is frequently lost in more aggressive, poorly differentiated melanomas (49)
. T-oligo also induced tyrosinase (Fig. 3C
), the rate-limiting enzyme in melanogenesis, shown to be induced by other T-oligos in normal human melanocytes (33
, 35)
and in human and murine melanoma cells (33
, 35
, 46)
and to be transcriptionally up-regulated by p53 within this time frame (50)
. T-oligo up-regulated MART-1 (Fig. 3D
), a protein expressed in melanocytes and melanomas that is a recognized target for a subclass of lymphocytes in melanoma immunotherapy (30)
. To our knowledge, this is the first report of an agent that up-regulates MART-1 or gp 100 expression.
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To explore the possibility that T-oligos may reduce MM-AN tumorigenesis and metastasis in vivo, MM-AN cells were treated for 48 h with T-oligo, complementary (control) oligo, or diluent alone, then collected, washed, and placed in oligo-free medium. Cells treated for 48 h with the oligos or diluent alone were injected into the tail vein of SCID mice, using 106 viable cells per injection, with viability determined by Trypan blue exclusion. Forty days after the injections, control mice had lost 12% of initial body weight, showed limited movement, and appeared severely ill whereas T-oligo animals had increased their weight by 27% and appeared healthy (Fig. 4
A). The mice were killed, an autopsy performed, and the average number and size of macroscopic metastases in the brain, liver, pancreas, and bone were determined by visual inspection of the organs. In contrast to animals injected with control cells (Fig. 4B
), animals injected with T-oligo-treated cells were virtually tumor-free, with no detectable metastases in four of five mice. T-oligo treatment of MM-AN cells before injection reduced the average volume of the metastases by 80–85% compared with control groups (Fig. 4C
) and the average number of metastatic tumors was reduced by 90–95% (Fig. 4D
). Although pre-treatment of malignant cells in this model is not equivalent to treating metastases already established in vivo, these data demonstrate that relatively brief exposure to T-oligos can have a long-lasting effect on these cells that cannot be reversed by the tissue environment. Moreover, because oligonucleotides of this size with physiologic phosphodiester linkage have a half-life in culture of 4–6 h (51)
, the effective exposure time of MM-AN cells to T-oligos in these experiments is probably far less than the 48 h in vitro incubation period.
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To determine whether T-oligo can affect the growth of pre-existing tumors, 2 x 106 MM-AN cells were injected subcutaneously into the flank of SCID mice. Beginning 72 h later, when small tumor nodules were first apparent, the sites were injected twice daily with 60 nmol of T-oligo, complementary (control) oligo, or an equal volume of diluent alone. After 22 days, the animals were killed and the tumor size was determined. There was no significant difference in tumor size between diluent and control oligo-treated mice, but T-oligo inhibited tumor growth resulting in reduced tumor volume (P<0.05) by 84 and 88%, respectively, as determined by measurement of the tumors with calipers (Fig. 5
A); tumors were a clinically undetectable size in three of six mice (Fig. 5B
). As determined in tissue cross sections obtained 24 h after the last injections, T-oligo induced tumor cell apoptosis (Fig. 5C
) and greatly enhanced expression of the differentiation-associated proteins gp100 and TRP-1 in the small residual tumor nodules (Fig. 5D
). The tissue surrounding the injected tumor nodules appeared normal in all animals.
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To determine whether T-oligo given systemically can affect the growth of pre-existing tumors, 1.5 x 106 MM-AN cells were injected subcutaneously in the flank and IP in SCID mice. Beginning 72 h later, the animals were injected twice daily IP with T-oligo or complementary oligo (420 µg in 100 µL PBS) or diluent for 25 days, then killed, and tumor size was determined. Mice treated with T-oligo had dramatically smaller subcutaneous tumors (Fig. 6
A), a 90% loss of volume compared with diluent or complementary oligo-treated mice (Fig. 6B
), establishing that T-oligo is effective after systemic delivery as well as after local administration. T-oligo-treated mice showed an 83 to 85% reduction in the number of intraperitoneal tumors (Fig. 6C, D
). Histologic examination of the tumors in the flank and the peritoneal cavity revealed that, as after direct local injection, T-oligo therapy markedly up-regulated differentiation markers and TUNEL positivity in residual tumor cells (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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makes it unlikely that the potential for increased immunorecognition of melanoma cells played a role in these experiments, although this anticipated benefit of T-oligo treatment was not explicitly examined. The T-oligo effects persisted in vivo for at least 40 days after an initial 48 h exposure of MM-AN cells in vitro and were observed to a comparable degree in MM-AN cell tumors established in vivo and subsequently treated with local or systemic T-oligo injections.
The key to successful therapy of malignancy is selective toxicity of the therapeutic agent for tumor cells, with sparing of the patient’s normal cells. Classic cancer therapy targets all dividing cells indiscriminately, resulting in bone marrow suppression, mucositis, and other life-threatening side effects. Only recently have advances in our understanding of the molecular basis of malignancy begun to allow targeting of features unique to the malignant cells, with a resulting increased efficacy and reduction in toxicity (28
, 53)
. The present data suggest that T-oligos preferentially affect malignant cells, as indeed would be expected if they trigger cellular responses directed against damaged DNA (see discussion below).
The mechanism by which T-oligos affect cells is partially known. Earlier work in our laboratory has shown that T-oligos activate the ATM kinase, leading to modification of the p95/Nbs1 protein responsible for S-phase arrest of the cell cycle (31)
. In the presence of continuous mitogenic stimulation, a G1/G0 arrest is subsequently achieved, presumably through p53 and p21 (39)
. Acting through p53, p73, and/or other as yet undocumented signaling pathways, T-oligos induce a large number of gene products involved in cell cycle regulation, DNA repair, differentiation, and induction of senescence (31
, 32
, 37
38
39)
. Of particular interest, T-oligos up-regulate p16INK4A and p27/Kip1 (39)
, proteins whose expression has been noted to decrease during the progression from primary to metastatic melanoma (5)
. In the case of MM-AN cells, T-oligos down-regulate livin/ML-IAP an anti-apoptotic protein known to be responsible for resistance to chemotherapy in at least some melanoma lines (8
, 9)
. Little is known regarding the pathways that regulate livin, although the closely related IAP survivin is down-regulated by p53 (54)
. Given that MM-AN cells lack detectable p53 and that in this cell line p73 mediates T-oligo-induced apoptosis (34)
, the data suggest that livin may be negatively regulated by p73. p73 itself is known to be activated by c-Abl (55)
, which in turn is activated by ATM after DNA damage (56)
. The ability of T-oligos to act through p73 to induce apoptosis (34)
, possibly through down-regulation of livin/ML-IAP, is notable in that up-regulation of p73 appears to be associated with melanoma progression (5)
, and p53 function is often lost in metastatic melanoma (7)
, as in the present instance. The data thus suggest that T-oligos may remain effective against melanoma even when one or more of the principal pathways mediating response to conventional chemotherapeutic agents have been compromised.
T-oligos activate the same pathways in normal human cells as in immortalized and/or transformed cells, although the limited data now available suggest that the consequences of this signal transduction are more dramatic in malignant cells. In cultured normal human melanocytes, the 2-base T-oligo thymidine dinucleotide induces a transient S-phase arrest (46)
but does not induce apoptosis, and instead simply promotes differentiation, as measured by melanin production and expression of tyrosinase over 1 wk exposure (33
, 35
, 46)
. Similarly, when applied once or twice daily to guinea pig or mouse skin over 2 wk, 2- to 9-base T-oligos enhance epidermal pigmentation but have no other morphologic effects on the tissue (35
, 36)
. In the present study, the 11-base T-oligo dose that caused apoptosis in two-thirds of human melanoma cells in vitro caused no apoptosis of normal human melanocytes, but only a transient S-phase arrest. This is consistent with the apparent lack of effect of either locally or systemically administered T-oligo on perilesional skin observed in the present in vivo experiments despite widespread apoptosis of the melanoma cells.
The effects of T-oligos on cultured cells are quite similar and perhaps identical to the effects of telomere loop disruption achieved by sequestration of the telomere binding protein TRF2 (13
14
15
, 31
, 34
, 39)
. The fact that disruption of the telomere loop, which exposes the single-stranded TTAGGG tandem repeats of the 3' overhang, and exogenous oligonucleotides with this sequence both evoke DNA damage-like responses led us to hypothesize that exposure of the TTAGGG sequence normally occurs after telomere loop disruption by critical telomere shortening (replicative senescence) or acute DNA damage (13
14
15
, 31
, 34
, 39)
. Our present and earlier (published) experience with melanoma and other malignant cell lines allows the further speculation that high levels of expression of telomerase, known to bind the telomere overhang, might obscure it in the event of telomere loop disruption, impairing the ability of malignant cells to detect this signal after acute DNA damage (Fig. 7
). Providing such cells a substitute signal—specifically, oligonucleotides homologous to the 3' overhang—might allow them to respond to their pre-existing extensive DNA abnormalities by evoking multiple ATM- and p53/p73-mediated cancer therapeutic responses, as observed in the present studies.
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Our data suggest that T-oligos may offer a novel approach to treatment of melanoma and other malignancies. Moreover, T-oligos may provide a powerful tool for exploring mechanisms of malignant progression and chemotherapy resistance.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication February 25, 2004. Accepted for publication May 5, 2004.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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