Antisense oligonucleotide targeting eukaryotic translation initiation factor 4E reduces growth and enhances chemosensitivity of non-small-cell lung cancer cells.

Elevated levels of eukaryotic translation initiation factor 4E (eIF4E) enhance translation of many malignancy-related proteins, such as vascular endothelial growth factor (VEGF), c-Myc and osteopontin. In non-small-cell lung cancer (NSCLC), levels of eIF4E are significantly increased compared with normal lung tissue. Here, we used an antisense oligonucleotide (ASO) to inhibit the expression of eIF4E in NSCLC cell lines. eIF4E levels were significantly reduced in a dose-dependent manner in NSCLC cells treated with eIF4E-specific ASO (4EASO) compared with control ASO. Treatment of NSCLC cells with the 4EASO resulted in decreased cap-dependent complex formation, decreased cell proliferation and increased sensitivity to gemcitabine. At the molecular level, repression of eIF4E with ASO resulted in decreased expression of the oncogenic proteins VEGF, c-Myc and osteopontin, whereas expression of ß-actin was unaffected. Based on these findings, we conclude that eIF4E-silencing therapy alone or in conjunction with chemotherapy represents a promising approach deserving of further investigation in future NSCLC clinical trials.

Molecular pathways involved in the synergistic interaction of the PKC beta inhibitor enzastaurin with the antifolate pemetrexed in non-small cell lung cancer cells.

Conventional regimens have limited impact against non-small cell lung cancer (NSCLC). Current research is focusing on multiple pathways as potential targets, and this study investigated molecular mechanisms underlying the combination of the PKC beta inhibitor enzastaurin with the multitargeted antifolate pemetrexed in the NSCLC cells SW1573 and A549. Pharmacologic interaction was studied using the combination-index method, while cell cycle, apoptosis induction, VEGF secretion and ERK1/2 and Akt phosphorylation were studied by flow cytometry and ELISAs. Reverse transcription-PCR, western blot and activity assays were performed to assess whether enzastaurin influenced thymidylate synthase (TS) and the expression of multiple targets involved in cancer signaling and cell cycle distribution. Enzastaurin-pemetrexed combination was highly synergistic and significantly increased apoptosis. Enzastaurin reduced both phosphoCdc25C, resulting in G2/M checkpoint abrogation and apoptosis induction in pemetrexed-damaged cells, and GSK3 beta and Akt phosphorylation, which was additionally reduced by drug combination (-58% in A549). Enzastaurin also significantly reduced pemetrexed-induced upregulation of TS expression, possibly through E2F-1 reduction, whereas the combination decreased TS in situ activity (>50% in both cell lines) and VEGF secretion. The effects of enzastaurin on signaling pathways involved in cell cycle control, apoptosis and angiogenesis, as well as on the expression of genes involved in pemetrexed activity provide a strong experimental basis to their evaluation as pharmacodynamic markers in clinical trials of enzastaurin-pemetrexed combination in NSCLC patients.

Expression of group IIa secretory phospholipase A2 increases with prostate tumor grade.

PURPOSE: Arachidonate release contributes to prostate tumor progression as arachidonate is metabolized into prostaglandins and leukotrienes, potent mediators of immune suppression, cellular proliferation, tumor motility, and invasion. The group IIa sPLA2 (sPLA2-IIa) can facilitate arachidonate release from cellular phospholipids. We therefore sought to determine whether sPLA2-IIa expression might be related to the development or progression of prostatic adenocarcinoma (CaP). EXPERIMENTAL DESIGN: sPLA2-IIa expression was examined by Western blot analyses of CaP cells and xenografts and by immunohistochemistry of benign prostatic hyperplasias and primary human CaPs (n = 101) using a sPLA2-IIa-specific polyclonal antibody. RESULTS: sPLA2-IIa expression was increased dramatically in the androgen-independent CWR-22R and LNAI CaP cells versus the androgen-dependent CWR-22 and LNCaP cells. Immunohistochemical analyses revealed that sPLA2-IIa expression was also significantly increased with CaP development and advancing disease (trend analysis; Pearson correlation coefficient, P = 0.016). High-grade CaPs showed intense, uniform staining for sPLA2-IIa that was significantly different from that in adjacent benign prostatic hyperplasias (Fisher's exact test, P = 0.021) or low-grade CaP (P = 0.013), both of which showed only focal or weak sPLA2-IIa staining. Further, uniform sPLA2-IIa expression was directly related to the increased proliferative index that typifies advancing disease (P = 0.001). Most significantly, enhanced sPLA2-IIa expression was inversely related to 5-year patient survival (P = 0.015). CONCLUSIONS: These data show that sPLA2-IIa expression increases with progression to androgen-independence and is highest in the most poorly-differentiated, highest-grade primary human CaP samples.

AKT-1, -2, and -3 are expressed in both normal and tumor tissues of the lung, breast, prostate, and colon.

PURPOSE: The AKT/PKB kinase controls many of the intracellular processes that are dysregulated in human cancer, including the suppression of apoptosis and anoikis and the induction of cell cycle progression. Three isoforms of AKT have been identified: AKT-1, -2, and -3. Selective up-regulation of AKT-3 RNA expression has been reported in hormone-independent breast and prostate cancer cell lines suggesting that AKT-3 expression may be increased with breast or prostate tumor progression. To determine whether AKT-3 RNA expression is selectively up-regulated in human cancers and whether the patterns of AKT RNA expression may change with tumor development, we examined AKT isoform expression by RT-PCR in human cancer cell lines, primary human cancers, and normal human tissues. EXPERIMENTAL DESIGN: AKT-1, -2, and -3 RNA expression was examined by RT-PCR. Because up-regulated AKT-3 expression has been implicated in human breast and prostate cancer progression, we also examined AKT-3 expression levels by semiquantitative RT-PCR using matched normal/tumor first-strand cDNA pairs from colon, breast, prostate, and lung cancers. RESULTS: Our data reveal that the overwhelming majority of both normal and tumor tissues express all three of the AKT isoforms. Moreover, semiquantitative RT-PCR of matched normal/tumor pairs confirmed similar AKT-3 RNA expression levels in both normal and tumor tissue. CONCLUSIONS: Our data show that both normal and tumor tissues express all three of the AKT isoforms and indicate that tumorigenesis does not involve a dramatic shift in the RNA expression patterns of the three AKT isoforms.

Integrin-linked kinase expression increases with prostate tumor grade.

PURPOSE: Integrin-linked kinase (ILK) overexpression can suppress anoikis, promote anchorage-independent cell cycle progression, and induce tumorigenesis and invasion. Inhibition of ILK in prostatic adenocarcinoma (CaP) cells elicits cell cycle arrest and induces apoptosis. Furthermore, ILK expression increases with androgen-independent progression of human CaP cell lines, suggesting that increased ILK expression may be associated with CaP progression. EXPERIMENTAL DESIGN: To assess whether ILK expression may be related to CaP development and/or progression, we have evaluated ILK expression by immunohistochemistry in 100 human prostate tissues. RESULTS: We show that ILK expression increases significantly with CaP progression. ILK immunostaining is specifically increased in high-grade, primary human CaP relative to adjacent benign prostatic hyperplasia (P < 0.001), benign prostatic hyperplasia from patients without cancer (P < 0.002), and low-grade CaP (P = 0.003). ILK overexpression is specifically associated with the increased proliferative index (P = 0.001) that typifies CaP progression. Strikingly, intense uniform ILK immunostaining was inversely related to 5-year patient survival (P = 0.004). CONCLUSIONS: ILK expression increases dramatically with CaP progression. ILK expression is also specifically related to the disproportionately increased proliferative index that contributes to the net gain of CaP cells during progression. Finally, enhanced ILK expression is inversely related to 5-year patient survival. These data therefore implicate increased ILK expression in prostate tumor progression.

Transforming growth factor-beta receptor type I gene is frequently mutated in ovarian carcinomas.

Ovarian carcinomas (OCs), particularly recurrent OCs, are frequently resistant to transforming growth factor (TGF)-beta-mediated growth inhibition. Mutations in the TGF-beta receptor type II (TbetaR-II) gene are only evident in a minority of OCs, suggesting that other alterations of the TGF-beta signaling pathway may be involved in OC. Using PCR, cold single-strand conformation polymorphism, and DNA sequencing, we now show that 33% of primary OCs (10 of 30) harbor somatic changes in exons 2, 3, 4, and 6 of the TGF-beta receptor I (TbetaR-I) gene. Most of the changes are missense mutations and clustered largely in the catalytic domain of the receptor kinase. Interestingly, seven additional cases (23.3%) showed heterozygous carriers of an allelic variant [a 9-nucleotide deletion, del(GGC)(3)] in exon 1 of the TbetaR-I gene. This is in contrast with 10.6% of del(GGC)(3) heterozygous carriers in a recent report of a large normal population (n = 735; B. Pasche et al., Cancer Res., 59: 5678-5682, 1999). These results indicate that TbetaR-I is frequently mutated in OC and suggest that resistance to TGF-beta-mediated growth inhibition may frequently involve alterations of the TbetaR-I gene.

Aberrant methylation of the estrogen receptor and E-cadherin 5' CpG islands increases with malignant progression in human breast cancer.

Loss of expression for both the estrogen receptor-alpha and E-cadherin genes has been linked to disease progression in human ductal breast carcinomas and has been associated with aberrant 5' CpG island methylation. To assess when, during malignant progression, such methylation begins and whether such methylation increases with advancing disease, we have surveyed 111 ductal carcinomas of the breast for aberrant methylation of the estrogen receptor-alpha and E-cadherin 5' CpG islands. Hypermethylation of either CpG island was evident prior to invasion in approximately 30% of ductal carcinoma in situ lesions and increased significantly to nearly 60% in metastatic lesions. Coincident methylation of both CpG islands also increased significantly from approximately 20% in ductal carcinoma in situ to nearly 50% in metastatic lesions. Furthermore, in all cases, the pattern of methylation displayed substantial heterogeneity, reflecting the well-established, heterogeneous loss of expression for these genes in ductal carcinomas of the breast.

Translational control of malignancy: the mRNA cap-binding protein, eIF-4E, as a central regulator of tumor formation, growth, invasion and metastasis.

Recent studies have implicated the mRNA cap-binding protein, eIF-4E, as a key regulator of malignant progression. Indeed, the major intracellular signaling pathways involved in tumor growth and malignancy, the MAP kinase and PI3 kinase pathways, induce eIF-4E activity. Furthermore, immunohistochemical analyses have revealed that eIF-4E is overexpressed and related to disease progression in human cancers of the colon, head and neck, and breast. In experimental tumors, manipulation of eIF-4E function profoundly affects not only tumorigenesis but also tumor invasion and metastasis. While increasing global protein synthesis rates, the increased activity of eIF-4E that typifies both human and experimental tumors disproportionately enhances the translation of a specific array of potent growth regulatory and malignancy-related proteins, including c-myc, cyclin D1, ornithine decarboxylase, vascular endothelial growth factor, basic fibroblast growth factor and others. Herein, we review the data supporting the notion that, by coordinately upregulating the translation of numerous malignancy-related proteins, eIF-4E plays a pivotal role in regulating not only tumor growth, but also invasion and metastasis.

Increased AKT activity contributes to prostate cancer progression by dramatically accelerating prostate tumor growth and diminishing p27Kip1 expression.

The PTEN tumor suppressor gene is frequently inactivated in human prostate cancers, particularly in more advanced cancers, suggesting that the AKT/protein kinase B (PKB) kinase, which is negatively regulated by PTEN, may be involved in human prostate cancer progression. We now show that AKT activation and activity are markedly increased in androgen-independent, prostate-specific antigen-positive prostate cancer cells (LNAI cells) established from xenograft tumors of the androgen-dependent LNCaP cell line. These LNAI cells show increased expression of integrin-linked kinase, which is putatively responsible for AKT activation/Ser-473 phosphorylation, as well as for increased phosphorylation of the AKT target protein, BAD. Furthermore, expression of the p27(Kip1) cell cycle regulator was diminished in LNAI cells, consistent with the notion that AKT directly inhibits AFX/Forkhead-mediated transcription of p27(Kip1). To assess directly the impact of increased AKT activity on prostate cancer progression, an activated hAKT1 mutant was overexpressed in LNCaP cells, resulting in a 6-fold increase in xenograft tumor growth. Like LNAI cells, these transfectants showed dramatically reduced p27(Kip1) expression. Together, these data implicate increased AKT activity in prostate tumor progression and androgen independence and suggest that diminished p27(Kip1) expression, which has been repeatedly associated with prostate cancer progression, may be a consequence of increased AKT activity.

Methylation patterns of the E-cadherin 5' CpG island are unstable and reflect the dynamic, heterogeneous loss of E-cadherin expression during metastatic progression.

Metastatic progression of most common epithelial tumors involves a heterogeneous, transient loss of expression of the homotypic cell adhesion protein, E-cadherin, rather than the uniform loss of a functional protein resulting from coding region mutation. Indeed, whereas E-cadherin loss may promote invasion, reexpression may facilitate cell survival within metastatic deposits. The mechanisms underlying such plasticity are unclear. We now show that the heterogeneous loss of E-cadherin expression in primary human breast cancers reflects a heterogeneous pattern of promoter region methylation, which begins early prior to invasion. In cultured human tumor cells, such heterogeneous methylation is dynamic, varying from allele to allele and shifting in relation to the tumor microenvironment. Following invasion in vitro, which favors diminished E-cadherin expression, the density of promoter methylation markedly increased. When these cells were cultured as spheroids, which requires homotypic cell adhesion, promoter methylation decreased dramatically, and E-cadherin was reexpressed. These data show that the methylation associated with E-cadherin loss in human breast cancer is heterogeneous and unstable and suggest that such epigenetic plasticity may contribute to the dynamic, phenotypic heterogeneity that drives metastatic progression.

Roles of trk family neurotrophin receptors in medullary thyroid carcinoma development and progression.

Although initiating mutations in the ret protooncogene have been found in familial and sporadic medullary thyroid carcinoma (MTC), the molecular events underlying subsequent tumor progression stages are unknown. We now report that changes in trk family neurotrophin receptor expression appear to be involved in both preneoplastic thyroid C cell hyperplasia and later tumor progression. Only a subset of normal C cells expresses trk family receptors, but, in C cell hyperplasia, the affected cells consistently express trkB, with variable expression of trkA and trkC. In later stages of gross MTC tumors, trkB expression was substantially reduced, while trkC expression was increased and often intense. In a cell culture model of MTC, exogenous trkB expression resulted in severely impaired tumorigenicity and was associated with 11-fold lower levels of the angiogenesis factor vascular endothelial growth factor. These results suggest that trk family receptor genes participate in MTC development and progression, and, in particular, that trkB may limit MTC tumor growth by inhibition of angiogenesis.

Deletion of p16INK4A/CDKN2 and p15INK4B in human somatic cell hybrids and hybrid-derived tumors.

Deletion or epigenetic inactivation of the tumor suppressor gene p16INK4/CDKN2 (p16) has been observed in multiple human tumors. We assayed hybrid cell lines between human diploid fibroblasts and fibrosarcoma cells for p16 allelic status and expression and found that p16 was expressed in the parental diploid fibroblast cell lines used, whereas the parental fibrosarcoma cell line HT1080.6TG exhibited homozygous deletion of p16. Most immortalized hybrid cell lines derived from these parent cell lines, whether tumorigenic or nontumorigenic, exhibited loss of fibroblast-derived p16 alleles. All p16-negative hybrid cell lines also exhibited deletion of p15INK4B (p15). Hybrid cell lines yielded tumors upon s.c. injection into athymic nude mice regardless of p16/p15 status. Tumors derived from six p16/p15-positive hybrid cells, however, revealed deletions of both p16 and p15. When human diploid fibroblasts were fused with A388.6TG squamous cell carcinoma cells, which exhibit aberrant methylation of p16, the resulting hybrids again exhibited deletion of the unmethylated fibroblast-derived p16 alleles. Transfection of both HT1080.6TG and A388.6TG cells with wild-type p16 expression vector resulted in decreased clonogenicity in culture. Although the determinants directing genetic versus epigenetic inactivation of p16 and p15 remain unclear, these results demonstrate that p16-mediated growth suppression could be abrogated by either mechanism in somatic cell hybrids.

Methylation-associated silencing of the tissue inhibitor of metalloproteinase-3 gene suggest a suppressor role in kidney, brain, and other human cancers.

Tissue inhibitor of metalloproteinase-3 (TIMP-3) antagonizes matrix metalloproteinase activity and can suppress tumor growth, angiogenesis, invasion, and metastasis. Loss of TIMP-3 has been related to the acquisition of tumorigenesis. Herein, we show that TIMP-3 is silenced in association with aberrant promoter-region methylation in cell lines derived from human cancers. TIMP-3 expression was restored after 5-aza-2'deoxycytidine-mediated demethylation of the TIMP-3 proximal promoter region. Genomic bisulfite sequencing revealed that TIMP-3 silencing was related to the overall density of methylation and that discrete regions within the TIMP-3 CpG island may be important for the silencing of this gene. Aberrant methylation of TIMP-3 occurred in primary cancers of the kidney, brain, colon, breast, and lung, but not in any of 41 normal tissue samples. The most frequent TIMP-3 methylation was found in renal cancers, which originate in the tissue that normally expresses the highest TIMP-3 levels. This methylation correlated with a lack of detectable TIMP-3 protein in these tumors. Together, these data show that methylation-associated inactivation of TIMP-3 is frequent in many human tumors.

Hypomethylation of pericentromeric DNA in breast adenocarcinomas.

Drug-induced DNA demethylation in normal human cells and inherited localized hypomethylation in mitogen-stimulated lymphocytes from patients with a rare recessive disease (ICF: immunodeficiency, centromeric region instability, facial anomalies) are associated with karyotypic instability. This chromosomal recombination is targeted to heterochromatin in the vicinity of the centromere (pericentromeric region) of human chromosome 1. Pericentromeric rearrangements in this chromosome as well as overall genomic hypomethylation are frequently observed in many kinds of cancer, including breast adenocarcinoma. We found that almost half of 25 examined breast adenocarcinomas exhibited hypomethylation in satellite 2 DNA, which is located in the long region of heterochromatin adjacent to the centromere of chromosome 1 and is normally highly methylated. One of the 19 examined non-malignant breast tissues displaying fibrocystic changes was similarly hypomethylated in this satellite DNA. We also looked at an opposing type of methylation alteration in these cancers, namely, hypermethylation in a tumor-suppressor gene region that is frequently hypermethylated in breast cancers. We found that increased methylation in the E-cadherin promoter region and decreased methylation in satellite 2 DNA were often present in the same breast cancers. While hypermethylation in certain tumor-suppressor gene regions may favor tumorigenesis by repressing transcription, demethylation of other DNA sequences may predispose to cancer-promoting chromosomal re-arrangements.

Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma.

Inactivation of the genes involved in DNA mismatch repair is associated with microsatellite instability (MSI) in colorectal cancer. We report that hypermethylation of the 5' CpG island of hMLH1 is found in the majority of sporadic primary colorectal cancers with MSI, and that this methylation was often, but not invariably, associated with loss of hMLH1 protein expression. Such methylation also occurred, but was less common, in MSI- tumors, as well as in MSI+ tumors with known mutations of a mismatch repair gene (MMR). No hypermethylation of hMSH2 was found. Hypermethylation of colorectal cancer cell lines with MSI also was frequently observed, and in such cases, reversal of the methylation with 5-aza-2'-deoxycytidine not only resulted in reexpression of hMLH1 protein, but also in restoration of the MMR capacity in MMR-deficient cell lines. Our results suggest that microsatellite instability in sporadic colorectal cancer often results from epigenetic inactivation of hMLH1 in association with DNA methylation.

Distinct patterns of E-cadherin CpG island methylation in papillary, follicular, Hurthle's cell, and poorly differentiated human thyroid carcinoma.

Expression of the invasion/metastasis suppressor, E-cadherin, is diminished or lost in thyroid carcinomas. Yet, mutational inactivation of E-cadherin is rare. Herein, we show that this loss is associated with hypermethylation of the E-cadherin 5' CpG island in a panel of human thyroid cancer cell lines. This aberrant methylation is evident in 83% of papillary thyroid carcinoma, 11% of follicular thyroid carcinoma, 40% of Hurthle's cell carcinoma, and 21% of poorly differentiated thyroid carcinomas. Contrary to previous reports, the majority of these poorly differentiated thyroid carcinomas express E-cadherin, but often within the cytoplasm rather than at the cell surface. Together, our data indicate that the invasion/metastasis suppressor function of E-cadherin is frequently compromised in human papillary, Hurthle's cell, and poorly differentiated thyroid carcinoma by epigenetic and biochemical events.

Alterations in DNA methylation: a fundamental aspect of neoplasia.

Neoplastic cells simultaneously harbor widespread genomic hypomethylation, more regional areas of hypermethylation, and increased DNA-methyltransferase (DNA-MTase) activity. Each component of this "methylation imbalance" may fundamentally contribute to tumor progression. The precise role of the hypomethylation is unclear, but this change may well be involved in the widespread chromosomal alterations in tumor cells. A main target of the regional hypermethylation are normally unmethylated CpG islands located in gene promoter regions. This hypermethylation correlates with transcriptional repression that can serve as an alternative to coding region mutations for inactivation of tumor suppressor genes, including p16, p15, VHL, and E-cad. Each gene can be partially reactivated by demethylation, and the selective advantage for loss of gene function is identical to that seen for loss by classic mutations. How abnormal methylation, in general, and hypermethylation, in particular, evolve during tumorigenesis are just beginning to be defined. Normally, unmethylated CpG islands appear protected from dense methylation affecting immediate flanking regions. In neoplastic cells, this protection is lost, possibly by chronic exposure to increased DNA-MTase activity and/or disruption of local protective mechanisms. Hypermethylation of some genes appears to occur only after onset of neoplastic evolution, whereas others, including the estrogen receptor, become hypermethylated in normal cells during aging. This latter change may predispose to neoplasia because tumors frequently are hypermethylated for these same genes. A model is proposed wherein tumor progression results from episodic clonal expansion of heterogeneous cell populations driven by continuous interaction between these methylation abnormalities and classic genetic changes.

Translation of ODC mRNA and polyamine transport are suppressed in ras-transformed CREF cells by depleting translation initiation factor 4E.

Rapid tumor growth and metastasis require increased polyamine metabolism, which is coordinately regulated by ornithine decarboxylase (ODC) and the polyamine transporter. Both activities are stimulated by ras signalling and are dependent upon protein biosynthesis. T24ras oncogene expression in rat embryo fibroblasts (CREFT24) induces cellular transformation and malignancy, in part, by stimulating the rate-limiting translation initiation factor, eIF-4E. CREFT24 expressing antisense RNA to eIF-4E (AS4E) have markedly decreased tumor growth rates and metastatic capacity, without altered monolayer growth rates. Herein, we demonstrate that in AS4E, ODC is translationally suppressed resulting in decreased ODC activity. Additionally, exogenous polyamine uptake is suppressed in AS4E cells indicating that AS4E can neither generate nor import the polyamines necessary to support rapid tumor growth. These data provide evidence that eIF-4E is the link between ras-induced malignancy and increased polyamine metabolism and support the hypothesis that eIF-4E plays a pivotal role in mediating ras-induced malignancy.

Mapping patterns of CpG island methylation in normal and neoplastic cells implicates both upstream and downstream regions in de novo methylation.

Promoter region CpG island methylation is associated with tumor suppressor gene silencing in neoplasia. GenBank sequence analyses revealed that a number of CpG islands are juxtaposed to multiple Alu repeats, which have been proposed as "de novo methylation centers." These islands also contain multiple Sp1 elements located upstream and downstream of transcription start, which have been shown to protect CpG islands from methylation. We mapped the methylation patterns of the E-cadherin (E-cad) and von Hippel-Lindau (VHL) tumor suppressor gene CpG island regions in normal and neoplastic cells. Although unmethylated in normal tissue, these islands were embedded between densely methylated flanking regions containing multiple Alu repeats. These methylated flanks were segregated from the unmethylated, island CpG sites by Sp1-rich boundary regions. Finally, in human fibroblasts overexpressing DNA methyltransferase, de novo methylation of the E-cad CpG island initially involved sequences at both ends of the island and the adjacent, flanking regions and progressed with time to encompass the entire CpG island region. Together, these data suggest that boundaries exist at both ends of a CpG island to maintain the unmethylated state in normal tissue and that these boundaries may be progressively overridden, eliciting the de novo methylation associated with tumor suppressor gene silencing in neoplasia.

Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.

Precise mapping of DNA methylation patterns in CpG islands has become essential for understanding diverse biological processes such as the regulation of imprinted genes, X chromosome inactivation, and tumor suppressor gene silencing in human cancer. We describe a new method, MSP (methylation-specific PCR), which can rapidly assess the methylation status of virtually any group of CpG sites within a CpG island, independent of the use of methylation-sensitive restriction enzymes. This assay entails initial modification of DNA by sodium bisulfite, converting all unmethylated, but not methylated, cytosines to uracil, and subsequent amplification with primers specific for methylated versus unmethylated DNA. MSP requires only small quantities of DNA, is sensitive to 0.1% methylated alleles of a given CpG island locus, and can be performed on DNA extracted from paraffin-embedded samples. MSP eliminates the false positive results inherent to previous PCR-based approaches which relied on differential restriction enzyme cleavage to distinguish methylated from unmethylated DNA. In this study, we demonstrate the use of MSP to identify promoter region hypermethylation changes associated with transcriptional inactivation in four important tumor suppressor genes (p16, p15, E-cadherin, and von Hippel-Lindau) in human cancer.