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Cancer Prevention and Research Center, Department of Pharmaceutical Sciences, College of Pharmacy, Washington State University, Pullman, WA 99164-6510
3To whom correspondence should be addressed. E-mail: meadows{at}wsu.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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KEY WORDS: • neoplasm • amino acid • gene expression
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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). In fact, dietary Tyr and Phe restriction specifically inhibits metastasis of melanoma, leukemia, hepatocarcinoma and lung carcinoma in rodents (2
–4
). Restriction of these amino acids increases the response of murine melanoma to chemotherapeutic agents and prevents the development of drug resistance (5
, 6
). Additional mechanistic studies indicate that Tyr and Phe deprivation suppresses expression of the metastatic phenotype of melanoma and inhibits invasion of melanoma cells through reconstituted cell matrix (2
, 7
–9
).
The mechanisms underlying the decrease in invasion and metastasis during Tyr and Phe restriction are not completely understood. Deficiency of these amino acids may inhibit invasion and metastasis through their control of gene expression and function. Some genes contain amino acid response elements in their promotor regions (10
, 11
). Two of these genes that are important to tumor invasion and metastasis that respond to amino acid deprivation are the collagenase and tissue inhibitors of metalloproteinase genes (12
).
Recently, a number of metastasis suppressor genes have been identified (13
). One of these, mitogen-activated protein kinase kinase 4/stress-activated protein/Erk kinase (MKK4/SEK1),4
is a gene that suppresses metastasis of prostate cancer (14
). MKK4/SEK1 is also a signaling molecule that activates c-Jun NH2-terminal kinase (15
), and disruption of this gene leads to defective AP-1–dependent transcriptional activity (16
). Because Tyr and Phe deprivation increases binding of AP-1 to consensus oligonucleotides in an electrophoretic mobility shift assay (17
), it is likely that MKK4/SEK1 is modulated by Tyr and Phe deprivation. Thus, we examined the expression of this gene at the biochemical level in melanoma, breast and prostate cancer cells. Potential genes that are regulated by Tyr and Phe deprivation in vitro also were identified by differential display and suppression subtractive hybridization.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Human A375 melanoma cells, MDA-MB-231 breast cancer cells and DU-145 and PC-3 prostate cancer cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD). A375-SM, a highly metastatic variant of A375 cells, and human SB2 melanoma cells were obtained from Dr. Menashe Bar-Eli at The University of Texas MD Anderson Cancer Center, Houston, TX. Cells were routinely cultured in minimum essential medium (MEM, from Life Technologies, Grand Island, NY) supplemented with 10% (v/v) fetal bovine serum, 1 mmol/L sodium pyruvate, 2 mmol/L L-glutamine, 60 IU/mL penicillin, 100 IU/mL streptomycin and 1% (v/v) nonessential amino acids (Sigma, St. Louis, MO). Tyr- and Phe-free MEM was custom manufactured by Life Technologies. Cells were cultured in MEM until they became 40% confluent. Then, the medium was replaced by fresh MEM or Tyr- and Phe-free MEM. Samples were collected at different time intervals as appropriate and each experiment was repeated twice.
Western blot analysis.
Cells were lysed on ice in buffer containing 20 mmol/L Tris-HCl at pH 7.4, 150 mmol/L NaCl, 0.5% NP-40, 1% SDS, 1 mmol/L EDTA and a proteinase inhibitor mixture, sonicated and then centrifuged at 13,000 x g for 5 min. Equivalent amounts of protein from each cell lysate were boiled for 5 min, electrophoresed on SDS-polyacrylamide gels and then transferred onto nitrocellulose membranes. The membranes were incubated with primary antibody against rabbit phosphorylated MKK4/SEK1 (Cell Signaling Technology, Beverly, MA). The filter was probed a second time with rabbit anti-MKK4/SEK1 (Sigma). For controlling the protein loading, the amount of actin from each sample was also detected using goat antiactin antibody (Santa Cruz Biotechnology, Santa Cruz, CA). After being washed with PBS containing 0.075% Tween 20, the membrane was incubated with relevant secondary antibodies (DAKO, Carpinteria, CA). Membranes were treated with enhanced luminol reagents (NEN Life Sciences Products, Boston, MA) and exposed to X-ray film. The band density was analyzed with the UVP Bioimaging system Labworks 4.0 program (Upland, CA).
Total RNA isolation and mRNA preparation.
Total RNA was isolated from tumor cells with Trizol reagent (Life Technologies) according to the manufacturer’s instructions. mRNA was prepared with the Micro-FastTrack 2.0 isolation kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol.
Differential display.
RNA isolated from A375 melanoma cells cultured in complete medium or Tyr- and Phe-free medium for 2 d was used for differential display analysis. For each reaction, one of the three oligo-dT-anchored primers, dT12-A/C/G, was used to reverse transcribe 2 µg total RNA into cDNA using M-MLV reverse transcriptase (Promega, Madison, WI). Ten-mer arbitrary primers (Operan, Alameda, CA) and 
-33P-dATP were used for the polymerase chain reaction (PCR). PCR was performed with one cycle of predenaturation at 94°C for 5 min followed by 40 cycles of PCR at 94°C for 30 s, 42°C for 2 min and 72°C for 30 s. The final extension reaction was run at 72°C for 5 min. The PCR products were fractionated in a sequence gel and exposed to X-ray film. Differentially expressed bands were excised from the gel, and the DNA fragments were recovered. Each fragment was used as a template to run PCR using the same conditions as described above. PCR products were cloned into the pGEM-T Easy Vector System (Promega) and the inserts were sequenced. Each DNA sequence was used to search the Genbank database with BLAST. The full cDNA sequence was recovered by the rapid amplification of the cDNA ends (RACE) method. Only genes modulated fourfold or greater were selected for further analysis.
Suppression subtractive hybridization.
The PCR-Select cDNA subtraction kit (Clontech, Palo Alto, CA) was used according to manufacturer’s instructions to construct a subtractive cDNA library and to clone the genes differentially regulated by Tyr and Phe deprivation. mRNA (2 µg) from A375 cells cultured in complete medium or in Tyr- and Phe-free medium for 2 d was used. The PCR products were cloned into the pGEM-T Easy Vector System to construct a subtractive cDNA library. The cDNA library was screened by PCR and the PCR products were spotted onto nylon membranes to make the cDNA array. The cDNA array was hybridized with the biotin-labeled cDNA from A375 cells cultured in complete medium or from cells cultured in Tyr- and Phe-free medium. Differentially regulated genes were sequenced and the full cDNA sequences were recovered by RACE. Only the genes down-regulated by Tyr and Phe deprivation were sequenced.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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). Expression of the MKK4/SEK1 protein was not changed by Tyr and Phe deprivation in A375 melanoma cells; however, the phosphorylated form was increased by almost threefold within 24 h of deprivation. This was followed by a decrease to basal levels at 72 h (Fig. 1
A). In contrast, phosphorylation of MKK4/SEK1 was decreased after 24 h of Tyr and Phe deprivation in A375-SM melanoma cells (Fig. 1
B). In SB-2 melanoma cells, Tyr and Phe deprivation decreased MKK4/SEK1 protein after 48 h; however, phosphorylation of the protein did not change (Fig. 1
C). Expression of MKK4/SEK1 protein was not changed in PC-3 prostate cancer cells, but phosphorylation of MKK4/SEK1 protein decreased progressively from 48 to 72 h after deprivation of Tyr and Phe (Fig. 1
D). Interestingly, MKK4/SEK1 protein expression increased in response to Tyr and Phe deprivation in DU-145 prostate cancer and MDA-MB-231 breast cancer cells; however, the phosphorylated protein was not detectable in these cells (Fig. 1
E, F).
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Reverse transcriptase-PCR–based mRNA differential display and suppression subtractive hybridization were used to study the genes differentially regulated by Tyr and Phe deprivation. Our purpose was to study the genes related to tumor invasion and metastasis. Differential display analysis identified 14 genes regulated by Tyr and Phe deprivation. Among them, three unknown genes were identified whose sequence as well as function has not been reported in the Genbank database. The functions of three other differentially regulated genes have not been examined. The names and functions of these genes are shown in Tables 1
and 2.
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).
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–19
), gene transcription (20
), angiogenesis (21
) and tumor metastasis (22
, 23
). The gene for enolase and FUS/CHOP, two genes that were down-regulated in A375 melanoma cells by Tyr and Phe deprivation, have been used as diagnostic markers (24
, 25
). In conclusion, this study shows that MKK4/SEK1 protein expression and phosphorylation are modulated by deprivation of Tyr and Phe and that the response is heterogeneous among and within different human tumor cell lines. We also found that a number of genes related to tumor progression are modulated by Tyr and Phe deprivation. The expression and the function of these genes are currently being examined.
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FOOTNOTES |
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2 Supported by grants 01A022 from the American Institute for Cancer Research and CA77604 from the National Cancer Institute.
4 Abbreviations used: MEM, minimum essential medium; MKK4, mitogen-activated protein kinase kinase 4; PCR, polymerase chain reaction; RACE, rapid amplification of the cDNA ends; SEK1, stress-activated protein/Erk kinase.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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1. Norris, J. R., Meadows, G. G., Massey, L. K., Starkey, J. R., Sylvester, D. M. & Liu, S.-Y. (1990) Tyrosine- and phenylalanine-restricted formula diet augments immunocompetence in healthy humans. Am. J. Clin. Nutr. 51:188-196.
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17. Thomas, A. W., Majid, A., Sherratt, E. J., Gagg, J. W. & Alcolado, J. C. (1999) Differential expression of mRNA in human thyroid cells depleted of mitochondrial DNA by ethidium bromide treatment. Clin. Sci. (Lond.) 97:207-213.[Medline]
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