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1 Department of Oncological Sciences, Mount Sinai School of Medicine, New York, New York; and 2 Laboratory of Molecular Technology, NCI, Frederick, Maryland
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B (NF-B), have been implicated in melanoma development and progression (5, 6, 7, 8, 9)
.
ATF2 is a member of the bZIP family of transcription factors that requires heterodimerization with other members of this family, including c-Jun, JunD, JunB, Fos, Fra1, ATFx, and ATFa (10
, 11)
, as well as with other regulatory components, including NF-B and retinoblastoma (12
, 13)
, to elicit its transcriptional activities. Also prerequisite is its phosphorylation by JNK and p38 (14
, 15)
. Inhibition of ATF2 activities results in sensitization of melanoma as well as breast cancer cells to apoptosis after treatment with radiomimetic or chemotherapeutic drugs that by themselves fail to affect these tumors (16)
. Additionally, expression of a 50aa peptide obtained from the amino terminal domain of ATF2 suffices to inhibit ATF2 transcriptional activities and sensitize melanoma to treatment, as well as to inhibit tumor growth in vivo (17)
. This region was shown to be important for the association of JNK and p383
(18)
, which is prerequisite to their phosphorylation of ATF2 on aa 69 to 71; mutation within the residues required for JNK but not p38 association on ATF2 impairs the activities of the ATF2 peptide both in vitro and in vivo (18)
. Here we show that a 10aa peptide that contains the JNK binding site on ATF2 increases JNK association with c-Jun, resulting in concomitant increase in spontaneous apoptosis and inhibition of tumorigenicity of melanoma in vivo.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Constructs.
The short ATF2 peptides were generated by introducing stop codons at the respective position into hemagglutinin (HA)-penetratin pcDNA3 ATF2 51 to 100aa vector (16)
with the Quick Change Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). The 5xJun2-Luc and 5xTRE-Luc constructs have been described previously (16
, 17)
. DNA sequencing and reverse transcription-PCR were carried out in all of the cases to confirm the integrity of each construct as well as its expression levels. Amino acids 51 to 100 of ATF2 were cloned into the HIV-TAT construct (19)
within the NcoI and XhoI sites. The fusion protein was expressed in the BL21 (DE3) pLysS (Novagen, Madison, WI) bacterial strain, and proteins were induced after the standard isopropyl-1-thio-ß-D-galactopyranoside protocol before being subjected to purification with the aid of nickel-nitrilo–triacetic acid beads. The retroviral vector used to express green fluorescent protein (GFP) in SW1 cells is a derivative of the Moloney Murine Leukemia virus vector pMMP412 into which an internal ribosome entry site-puromycin resistance cassette was inserted downstream of the GFP.
Transcription Analysis.
Reporter constructs indicated in Results (0.3 µg) were cotransfected with the respective expression vectors into melanoma cells (5 x 105) with LipofectAMINE (Invitrogen, Carlsbad, CA). Luciferase activity was measured using the Luciferase assay system (Promega, Madison, WI).
Apoptosis Studies.
Cells were analyzed to detect spontaneous (basal) or induced apoptosis [after treatment with the kinase inhibitor UCN-01 (kindly provided by National Cancer Institute repository) for 36 hours]. Apoptosis was measured by using fluorescence-activated cell sorter (8)
to quantify the percentage of hypodiploid nuclei undergoing DNA fragmentation. Nucleation as a marker of apoptosis was monitored via 4',6-diamidino-2-phenylindole staining of cells at indicated time points after their treatment. Caspase activity was monitored by immunoblots with antibodies to caspase 9 (Santa Cruz Biotechnology, Santa Cruz, CA). Poly(ADP-ribose) polymerase (PARP) cleavage was monitored by corresponding antibodies (PharMingen, San Diego, CA).
In Vitro Binding Assay.
Flag JNK2 expressed in 293T cells extracted, immunopurified with anti-Flag antibody bound to protein G agarose beads were first incubated with 3 mg/mL of BSA in PBS for 2 hours, followed by incubation at 4°C for 2 hours with 35S-labeled in vitro translated c-Jun or ATF2 (TNT-coupled reticulocyte lysate system, Promega) in the presence of wild-type or mutant peptide. Bead-bound material was subjected to three washes with 20 mmol/L Tris (pH 7.5), 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 0.5% NP40, 1 mmol/L NaVO4, and 1 mmol/L DTT supplemented with protease inhibitors. Reaction mixtures were then separated on SDS-PAGE and transferred onto a nitrocellulose membrane. Binding was detected by autoradiography and quantified with the aid of a phosphorimager. The wild-type and the mutant ATF2 51 to 60aa peptides were synthesized (Sigma Genosys, Woodland, TX) at a scale of 5 mg with >95% purity.
Tumor Growth in Vivo.
LU1205, FEMX, and SW-1 cells that express control or ATF251–100 peptide were trypsinized, resuspended in PBS, and injected s.c. (1 x 106) into 6- to 7-week–old mice in the lower flank. When the tumor reached the size of about 50 mm3, 10 µmol/L of SB203580 was injected into four of the LU1205 tumors every 4 days during a 2-week period. For FEMX tumors, UCN-01 (5 mg/kg) was fed by gavage three times per week for a total of 2 weeks. Tumor growth was monitored every two days. SW-1 cells that express wild-type or mutant forms of the ATF2 peptide were trypsinized, resuspended in PBS, and injected subcutaneously in the lower flank (1 x 104) of 6- to 7-week-old mice as described previously (17)
. GFP-expressing SW1 tumors were monitored in vivo on shaved mice at the indicated time points with a light box illuminated by blue light fiber optics (Lightools Research, Encinitas, CA), and imaging was carried out with a digital camera (Nikon D100). Injection of HIV-TAT, control or fused with ATF2 peptide, was performed on 48-mm3 size tumors at the indicated time points. Each injection was directed at multiple sites within the tumors. Tissue samples were fixed in formalin and embedded in paraffin. H&E terminal deoxynucleotidyl transferase (Tdt)-mediated nick-end labeling (TUNEL) staining was performed as described previously (16)
.
The cDNA Microarray Hybridization.
The 10k mouse Gem 2 gene set (Incyte Genomics Inc., Palo Alto, CA) was printed at the National Cancer Institute on poly-L–lysine-coated glass with a Biorobotics TASII arrayer (Cambridge, England). All protocols for manufacturing and hybridization of microarrays are posted on the National Cancer Institute web site (http://nciarray.nci.nih.gov). Approximately 20 µg of total RNA were used in the reverse transcription reaction to directly label the probe with Cy-5 dUTP or Cy-3 dUTP (Amersham, Piscataway, NJ). Hybridizations were performed at 65°C for 12 to 18 hours in a hybridization volume of 35 µL. The hybridized arrays were scanned with an Axon GenePix 4000 scanner (Union City, CA), and fluorescence data were collected with the GenePix software.
Data Analysis.
The normalization of the sample intensities and calculation of the calibrated fluorescence ratios after background subtraction were determined with the GenePix 4.0 software package (Axon Instrument, Union City, CA). Four independent array hybridizations were conducted for each sample. Spots that did not pass predefined criteria of minimum signal to noise were excluded. Filters were set to eliminate any spots for which the signals were <2-fold higher than the normalized background. Signals because of the presence of localized artifacts on the array image such as dust or lint were flagged as "bad" and excluded from the dataset. The ratio distribution extracted from each microarray image exhibited normal distribution, constant coefficient-of-variation, and high positive signal (Fig. 5)
. We used 2-fold above background as the intensity cutoff in all of the experiments. Thus, satisfying the statistical conditions required to determine the significance of ratio measurements by the confidence interval generated with web-based microarray analysis tools at mAdb. Under our experimental conditions, a 99% confidence interval defined ratios of 2 or larger or 0.5 and smaller as indicators of substantially different gene expression levels between two samples hybridized to the same array spot. We have used expression ratio calculations to determine whether gene expression differs substantially between the red and green channels. The distribution parameter can be estimated with a maximum likelihood method, and ratio calibration can be carried out by an iterative method. In our analyses, we have found that ratios of >2.0 are consistent on repeated hybridizations, although values as low as 1.5 were often found to be substantial after real-time PCR validation.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. An additional decrease in growth of tumors that express ATF251–100 was found when they were treated with the pharmacological inhibitor of p38 or the chemotherapeutic drug UCN-01 (Fig. 1, A and B)
. TUNEL analysis revealed a marked increase in the degree of apoptosis in tumors expressing the ATF251–100 peptide on their exposure to additional treatment (Fig. 1C)
. These results suggest that expression of the ATF251–100 peptide suffices to slow the growth rate and to sensitize human melanoma tumors to apoptosis following treatment.
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Injection of HIV-TAT–ATF251–100 Inhibits Growth and Metastasis of SW1 Tumors.
Because expression of the ATF2 peptide (constitutive, inducible, or via adenovirus delivery) was shown to efficiently inhibit the formation of SW1 as well as B16F10 tumors in vivo (17)
, we assessed whether administration of the peptide as a bacterially produced fusion protein would suffice to inhibit melanoma growth. To this end we chose to use the HIV-TAT system, given its ability to elicit potent delivery of its fusion proteins through cellular membranes (19)
. Further, all of the ATF2 peptide constructs contain a fusion protein with penetratin, which resemble those of the HIV-TAT peptide (17)
. Following its production in bacteria and purification on nickel beads (Fig. 2A)
, 1 µg/mm3 of the peptide was injected into 48 mm3-size SW1 tumors followed by a second injection at the indicated time points (Fig. 2B)
; thus, the amount of peptide injected was directly proportional to the size of the tumor. Before their injection into mice, tumor cells used in this study were infected with GFP, enabling follow-up observations of tumor size in real time without need of surgery. Using a UV lamp allowed monitoring changes in tumor mass, as shown in Fig. 2B
. Whereas the control HIV-TAT construct had no effect on SW1 tumor growth, injection of the HIV-TAT–ATF251–100 fusion protein caused a marked decrease in tumor mass noticeable within a day or two after the first injection, and more so after 4 days (Fig. 2B)
. These data provided the first evaluation of the effect of ATF2 peptide in vivo at different stages after its administration. Further analysis was carried out upon termination of the experiment, 13 days after the second injection. Of 20 tumors 9 were no longer visible after 3 days (2 tumors), 6 days (4 tumors), and 10 days (3 tumors) after the first injection of the HIV-TAT–ATF251–100 peptide. At the end of the experiment, 16 days after the first injection of the peptide, 11 of 20 tumors exhibited marked growth inhibition (Fig. 2C)
.
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. Microscopic examination using H&E staining confirmed the presence of multiple metastatic lesions within the lungs of control animals but not in the ATF251–100 peptide-expressing group (Fig. 3B)
. Lung metastases were substantially more pronounced upon visualization of GFP under a fluorescence microscope, which enabled detection of GFP-positive lesions (Fig. 3C)
. These findings confirm that the GFP-expressing SW1 cells are those that metastasized to generate these lesions. Significantly, no such lesions were found in animals into which the HIV-TAT–ATF251–100 peptide had been injected (Fig. 3C)
. Together, these data suggest that administration of the ATF251–100 peptide as a HIV-TAT fusion peptide into tumors results in efficient inhibition of tumorigenesis and metastasis of these otherwise aggressive tumors, similar to what was observed upon the delivery of peptide by adenovirus or constitutive or inducible expression (17)
.
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. Subcutaneous injection of 106 SW1 cells expressing the control construct resulted in formation of 1,100-mm3 size tumors within 18 days. However, expression of the 50aa peptide in these cells (constitutive expression based on selection of drug-resistant cells) decreased tumor size to 300 mm3, a result that resembled the degree of inhibition in previous studies (17)
. Strikingly, constitutive expression of each of the 3 shorter peptides also elicited inhibition of melanoma growth that varied from 400 to 600 mm3 (Fig. 4B)
. Both the 20aa and 10aa peptides, spanning aa 51 to 60 and aa 51 to 70, respectively, elicited efficient inhibition close to that observed with the 50aa peptide. These data suggest that the 51 to 60aa peptide harbors the critical domain required for inhibition of SW1 growth in vivo. Given our intention to minimize the size of the peptide, additional evaluation was performed on the 10aa peptide.
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). Similarly, expression of ATF251–60 was also efficient in increasing TRE-Luc activities (Fig. 5A)
. These findings suggest that the short peptide elicits similar changes in promoters containing TRE sequences. In contrast, transcriptional activities from Jun2-Luc, which was efficiently inhibited upon expression of ATF251–100 (ref. 17
; Fig. 5B
), were no longer inhibited but slightly elevated in response to expression of the 51 to 60aa peptide. This suggests that inhibition of ATF2 transcriptional activities via the 50aa peptide could no longer be mediated in response to expression of the shorter peptide. These findings suggest that the 10aa peptide affects primarily the Jun/JNK signaling cascade via altered TRE-dependent activities.
Expression of ATF251–60 Induces Spontaneous Apoptosis of Melanoma Cells.
We next assessed the degree of apoptosis in SW1 cells under normal growth (spontaneous) as well as after treatment with chemotherapeutic drugs. Normally, SW1 cells are highly resistant to apoptosis after various treatments (16
, 17)
. Such resistance was reduced upon expression of the ATF251–60 peptide, because treatment with the protein kinase inhibitor UCN-01 (shown as representative of various treatments) induced an increase (20 to 40%) in apoptosis (Fig. 5C)
. However, the degree of UCN-01–induced apoptosis was substantially higher in melanoma cells that express the 50aa peptide (20 to 77%; Fig. 5C
). This observation suggests that the 10aa peptide is less efficient in sensitization of melanoma cells to apoptosis after treatment.
However, SW1 cells that express the shorter peptide underwent spontaneous apoptosis, which was seen in the absence of treatment (12 to 37%; 
, third panel, Fig. 5C
). Induction of basal apoptosis was also seen, at somewhat higher levels, upon expression of the ATF251–100 peptide (12 to 55%; , second panel, Fig. 5C
). These findings suggest that whereas sensitization by the full-length peptide (aa 51 to 100) is mediated via two distinct mechanisms–spontaneous and inducible apoptosis–the shorter peptide51–60 primarily elicits spontaneous, and is less efficient in inducing, apoptosis in response to treatment.
The ATF2 Peptides Activate Caspase 9 and Causes PARP Cleavage.
To assess which of the proapoptotic components is affected upon expression of ATF2 peptides, we have analyzed possible changes in the profile of caspases. As shown in Fig. 6
, treatment of both human and mouse melanoma cells with UCN-01 caused activation of caspase 9, as reflected by the formation of the corresponding cleavage fragments. Cells that express either the 10 or 50aa peptides revealed activation of caspase 9 even before treatment with UCN-01. Treatment with UCN-01 retained the same level of caspase 9 cleavage as seen before treatment (Fig. 6)
. These data indicate that the expression of the ATF2 peptides is sufficient for the activation of caspase 9, which could explain, in part, how expression of these peptide causes spontaneous apoptosis. Similarly, PARP cleavage was observed upon treatment of the control cells, but also in cells that express ATF2 peptides, before their exposure to UCN-01 (Fig. 6)
. Indeed, caspase 3, which is responsible for PARP cleavage, was also activated on expression of the ATF2 peptides (data not shown). These data suggests that both peptides suffice to induce caspase and PARP cleavage, which reflects cell commitment for apoptosis.
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. Such an effect was not observed in JNK-ATF2 association (Fig. 7, A–C)
. This finding indicates that the ATF251–60 peptide efficiently increases the affinity of JNK for c-Jun, enabling greater phosphorylation and activation, which coincide with higher stability. Along these lines, mutation within the JNK binding region of the 50aa peptide markedly reduced its ability to inhibit tumorigenicity (18)
.
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. The mRNA was prepared from a pool of four tumors obtained from two different experiments. In both cases tumors were excised 2 weeks after subcutaneous injection of cells. Because the fluorescently labeled Cy dyes may incorporate into the reverse-transcribed cDNA with different efficiencies, RNA isolated from both cell types was always labeled with both combinations of Cy dyes. Genes either up- or down-regulated by >2.5-fold were identified on the basis of a scatter plot analysis (Fig. 8A)
. SW1 vector control cells were labeled with Cy-3 (F532), and the SW1-ATF2 51 to 100 peptide-expressing cells were labeled with Cy-5 (F635). Genes up-regulated in response to the expression of the ATF251–100 peptide appear red on the microarray, whereas genes down-regulated in tumors expressing the peptide (compared with control tumors) appear green (Fig. 8A)
. Clustering analysis of the array data revealed several interesting patterns of gene expression. A large group of tumor necrosis factor-related genes were strongly up-regulated in tumors that express the ATF251–100 peptide (Fig. 8B)
.
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. Down-regulation of Fas-associated genes would result in reduced apoptosis via the Fas pathway and explain why there was no change in the growth and development of these tumors in GLD mice, which are deficient in Fas ligand (ref. 17
; data not shown). Concomitant with decrease in Fas-associated genes was up-regulation of tumor necrosis factor-related transcripts, which constitutes the other major apoptosis cascade. These changes reveal that expression of the ATF2 peptide efficiently altered the balance of apoptosis cascades from Fas toward the tumor necrosis factor pathway, which otherwise provides a promitogenic signal in late stage melanomas (20)
. Interestingly, we also observed a consistent down-regulation of growth-associated genes including epidermal growth factor, hepatoma-derived growth factor, and insulin-like growth factor, and several interferon-associated genes (Fig. 8B)
.
Among other up-regulated transcripts (Table 1A)
were insulin-like growth factor binding protein 2 (implicated in insulin-like growth factor activity, which has been associated with radio resistance and apoptosis; refs. 21
, 22
), interleukin 1ß (implicated in inhibition of angiogenesis and metastasis of melanoma and other tumors; ref. 23
), cullin 3 (ubiquitin protein ligase that controls cyclin E in ubiquitination and consequently regulates entry into the S phase; refs. 24
, 25
), kinesin-associated protein 3 (a kinesin superfamily associated protein implicated in organelle transport; refs. 26
, 27
), ATF3 (which represses cyclic-AMP responsive element-dependent transcription and accelerates caspase protease activation during DNA damaging agent-induced apoptosis; ref. 28
), and membrane metalloendopeptidase (implicated in invasion and metastasis; ref. 29
).
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were tyrosine kinase 2, plasminogen activator (implicated in invasion and metastasis; ref. 30
), metallothionein 2 (which confers resistance to metals; ref. 31
), microtubule-associated protein myosin Vb (associated with the plasma membrane recycling system; ref. 32
), and ubiquitin-specific protease 18 (a type I interferon-inducible gene that contributes to growth arrest and differentiation in human melanoma cells treated with IFN-ß; ref. 33
). This panel provides important mechanistic insights with regard to changes in the expression pattern of genes that occurred in vivo in the course of inhibiting tumor growth in response to expression of the ATF251–100 peptide. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The sensitization of human melanomas to treatment by either the pharmacological inhibitor of p38 or by the chemotherapeutic drug UCN-01 points to an important advance in treatment of these tumors by reagents that otherwise do not affect this tumor type. Such sensitization can be attributed to changes in apoptotic cascade (switch toward the tumor necrosis factor-related apoptosis-inducing ligand pathway; 17
) combined with decreased NF-B antiapoptotic activities4
and the activation of caspase 9 and caspase 3, based on the cleavage of PARP. Significantly, both caspase activation and PARP cleavage were seen on expression of the ATF2 peptides, thereby substantiating their ability to induce spontaneous apoptosis without additional treatment. It is important to emphasize that our studies in the SW1 and B16F10 tumor settings were limited to the effect of the peptide alone as it was sufficient to elicit striking inhibition of these aggressive tumors. It is expected that the effects of the ATF2 peptide on tumor growth rate and metastasis would be improved by combining the peptide with other treatments, including inhibitors of stress kinases, immunologic modulators, or chemotherapeutic drugs.
The present study extends the finding that the ATF251–100 peptide is capable of inhibiting melanoma growth and metastasis. Here we show that administration of the 50aa peptide as a fusion protein with HIV-TAT is sufficient to cause marked inhibition of the SW1 tumors, even to the point of their complete regression, while eliminating metastases commonly produced by tumors of this type.
Because of reduced ATF2 and elevated transcriptional activities of Jun family members, a large subset of genes regulated by ATF2 and c-Jun were found to exhibit altered expression–as shown here for elevated tumor necrosis factor-related genes and down-regulation of growth-associated and IFN-related genes, as revealed by the gene profiling analysis.
We additionally show that administration of a 10aa peptide that encompasses the JNK binding site sensitizes melanoma to spontaneous apoptosis in vitro and inhibits tumor formation in vivo. The short peptide preserves some of the activities characterized previously for the 51 to 100aa peptide, because it affects TRE- but not Jun-2-dependent transcription. As a result, tumors expressing this peptide are prone to undergo spontaneous apoptosis in the absence of external drugs or treatment. This sensitization is attributed to the change in TRE-mediated transcription, which is dependent on JNK-Jun and JunD (18) . The ability of the shorter peptide to alter TRE-dependent transcription is attributed to the ability of the peptide to increase JNK binding with its substrate, c-Jun. The greater association may have an impact on phosphorylation in trans of JunD (34) , as well as possible effects on other transcriptional regulators of Jun, including HDAC3, the repression of which Jun transcription depends on its phosphorylation by JNK (35) .
Equally intriguing is the finding that the shorter peptide did not affect Jun2 transcription, which coincides with the lower level of apoptosis induced upon treatment of cells that express the longer form of this peptide with chemotherapeutic drugs. This observation further highlights distinct mechanisms required for basal apoptosis versus those necessary for inducible apoptosis. Our findings also reveal the importance of basal apoptosis for inhibition of tumorigenicity of melanoma. Expression of the short peptide suffices for induction of spontaneous apoptosis and inhibition of melanoma growth in vivo. These observations are consistent with our earlier studies, which revealed that such inhibition is mediated in part by abundant apoptosis found in tumors expressing the ATF2 peptide (18) . These data, together with the ability of the HIV-TAT–ATF251–100 peptide to inhibit melanoma growth and metastasis, points to the possible use of these reagents for novel drug design in melanoma and possibly other tumor types.
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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: Ze’ev Ronai, Department of Oncological Sciences, Mount Sinai School of Medicine, Ruttenberg Cancer Center, New York, NY 10029. Fax: (212) 849-2425; E-mail: zeev.ronai{at}mssm.edu; RONAI{at}Burnham.org
3 N. Jones, personal communication. 
Received 2/27/04. Revised 8/31/04. Accepted 9/ 7/04.
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REFERENCES |
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