Cytokeratins in normal lung and lung carcinomas. I. Adenocarcinomas, squamous cell carcinomas and cultured cell lines

The various epithelial cells of the lower respiratory tract and the carcinomas derived from them differ markedly in their differentiation characteristics. Using immunofluorescence microscopy and two-dimensional gel electrophoresis of cytoskeletal proteins from microdissected tissues we have considered whether cytokeratin polypeptides can serve as markers of cell differentiation in epithelia from various parts of the human and bovine lower respiratory tract. In addition , we have compared these protein patterns with those found in the two commonest types of human lung carcinoma and in several cultured lung carcinoma cell lines. By immunofluorescence microscopy, broad spectrum antibodies to cytokeratins stain all epithelial cells of the respiratory tract, including basal, ciliated, goblet, and alveolar cells as well as all tumor cells of adenocarcinomas and squamous cell carcinomas. However, in contrast, selective cytokeratin antibodies reveal cell type-related differences. Basal cells of the bronchial epithelium react with antibodies raised against a specific epidermal keratin polypeptide but not with antibodies derived from cytokeratins characteristic of simple epithelia. When examined by two-dimensional gel electrophoresis, the alveolar cells of human lung show cytokeratin polypeptides typical of simple epithelia (nos. 7, 8, 18 and 19) whereas the bronchial epithelium expresses, in addition, basic cytokeratins (no. 5, small amounts of no. 6) as well as the acidic polypeptides nos. 15 and 17. Bovine alveolar cells also differ from cells of the tracheal epithelium by the absence of a basic cytokeratin polypeptide. All adenocarcinomas of the lung reveal a "simple-epithelium-type" cytokeratin pattern (nos. 7, 8, 18 and 19). In contrast, squamous cell carcinomas of the lung contain an unusual complexity of cytokeratins. We have consistently found polypeptides nos. 5, 6, 8, 13, 17, 18 and 19 and, in some cases, variable amounts of cytokeratins nos. 4, 14 and 15. Several established cell lines derived from human lung carcinomas (SK-LU-1, Calu -1, SK-MES-1 and A-549) show a uniform pattern of cytokeratin polypeptides (nos. 7, 8, 18 and 19), similar to that found in adenocarcinomas. In addition, vimentin filaments are produced in all the cell lines examined, except for SK-LU-1.(ABSTRACT TRUNCATED AT 400 WORDS)     

Occurrence of differentiated keratin peptide(K1) in cultured human squamous cell carcinomas.

To date, the largest keratin peptide(K1, 68 KD) has been absent in cultured human squamous cell carcinomas. Using a low salt aqueous solution, not containing high salt and Triton X-100, as a washing buffer for keratin extraction, followed by two dimensional polyacrylamide gel electrophoresis, immunological techniques and Northern blot analysis, we demonstrated K1 peptide in two kinds of cultured human squamous cell carcinomas. Until now keratin extraction has been done using high salt/Triton X-100 solution during which K1 peptide may be removed together developed an affinity with the buffer. Many investigators may have therefore overlooked K1.     

Subunit structure of the mouse epidermal keratin filament.

The two proteins which are the subunits of mouse epidermal keratin filaments have been isolated from fully differentiated epidermis (stratum corneum), viable differentiating cells and cells grown in culture. The proteins have molecular weights of 68 000 and 60 000, consist of families of very similar species, have common N-terminal (N-acetylserine) and C-terminal (glycine) residues, contain 35--40% alpha-helix and are immunologically cross-reacting. In mixtures, the two proteins polymerize in vitro into native-type keratin filaments that are 70--80 angstrom in diameter, up to 30 micrograms long, possess a characteristic alpha-type X-ray diffraction pattern and contain the subunits in the precise molar ratio of 1 : 2 or 2 : 1.     

Protein kinase activities in neoplastic squamous epithelia and normal epithelia from the upper aero-digestive tract

In the present study the activities of three different protein kinase were determined in squamous cell carcinoma from the upper aero-digestive tract, and compared with the activities in normal oral mucosa. The protein kinases investigated are: a) cAMP-dependent protein kinase; b) cGMP-dependent protein kinase, and c) casein kinase II. The basal protein kinase activity, when histone IIa was used as substrate, was about 3-fold higher in tumors, as compared to normal mucosa, in the soluble fraction (32.0 +/- 4.2 and 10.9 +/- 2.4 pmol 32P/mg prot. X min, respectively). In the particulate fraction the basal protein kinase activity was about 9 times higher in tumors as compared to normal mucosa (19.4 +/- 5.2 and 2.1 +/- 0.3 pmol 32P/mg prot X min, respectively). The protein kinase activity in the presence of cyclic nucleotide (cAMP/cGMP) minus the basal protein kinase activity was taken as the cAMP- and the cGMP-dependent protein kinase activity, respectively. Maximal protein kinase activity was obtained in the presence of 0.5 microM of cyclic nucleotide both in squamous cell carcinoma and normal mucosa. In the cytosolic fraction the cAMP-dependent protein kinase activity was 33.9 +/- 13.0 pmol 32P/mg prot. X min in tumors, and 28.2 +/- 5.8 pmol 32P/mg prot. X min in normal tissue, after stimulation with 0.5 microM cAMP. The cGMP-dependent protein kinase activity was 5-10% of the cAMP-dependent protein kinase activity, and no concentration-dependent stimulation with cGMP was seen. The cGMP-dependent protein kinase activity in the presence of 0.5 microM cGMP was 2.4 +/- 1.3 and 1.8 +/- 0.6 pmol 32P/mg prot. X min in tumors and normal mucosa, respectively. Casein kinase II activity was determined only in the cytosolic fraction and was found to be 3-fold higher in tumors as compared to normal mucosa (31.8 +/- 5.2 and 8.6 +/- 3.5 pmol 32P/mg prot X min, respectively). This study shows a general increase in histone phosphorylation and casein kinase activity in neoplastic squamous epithelia compared to normal epithelia. No evidence for an increase in cyclic nucleotide dependent protein kinase activities in neoplastic squamous epithelia was found. This study thus supports the idea that phosphorylation/dephosphorylation reactions may play an important role in the control of cell growth, differentiation and proliferation.     

Initiation of assembly of the cell envelope barrier structure of stratified squamous epithelia

The cell envelope (CE) is a specialized structure that is important for barrier function in terminally differentiated stratified squamous epithelia. The CE is formed inside the plasma membrane and becomes insoluble as a result of cross-linking of constituent proteins by isopeptide bonds formed by transglutaminases. To investigate the earliest stages of assembly of the CE, we have studied human epidermal keratinocytes induced to terminally differentiate in submerged liquid culture as a model system for epithelia in general. CEs were harvested from 2-, 3-, 5-, or 7-d cultured cells and examined by 1) immunogold electron microscopy using antibodies to known CE or other junctional proteins and 2) amino acid sequencing of cross-linked peptides derived by proteolysis of CEs. Our data document that CE assembly is initiated along the plasma membrane between desmosomes by head-to-tail and head-to-head cross-linking of involucrin to itself and to envoplakin and perhaps periplakin. Essentially only one lysine and two glutamine residues of involucrin and two glutamines of envoplakin were used initially. In CEs of 3-d cultured cells, involucrin, envoplakin, and small proline-rich proteins were physically located at desmosomes and had become cross-linked to desmoplakin, and in 5-d CEs, these three proteins had formed a continuous layer extending uniformly along the cell periphery. By this time >15 residues of involucrin were used for cross-linking. The CEs of 7-d cells contain significant amounts of the protein loricrin, typically expressed at a later stage of CE assembly. Together, these data stress the importance of juxtaposition of membranes, transglutaminases, and involucrin and envoplakin in the initiation of CE assembly of stratified squamous epithelia.     

Prostacyclin biosynthesis in activated, stimulated and normal mouse peritoneal cell populations.

Nonspecific resistance to infectious and neoplastic disease can be enhanced by administration of "immunomodulators". The levels of enhancement can be monitored by following in vitro function of cells of the lympho-reticuloendothelial system. To gain a better understanding of the physiological and biochemical nature of this enhancement, the metabolism of prostaglandin endoperoxide PGH2 was followed in mouse peritoneal cells (PCs). Homogenates of PCs from normal, unstimulated mice yielded primarily prostacyclin (PGI2) when incubated with PGH2. Homogenates of PCs from mice injected with the immunomodulators C. parvum, levamisole HCl, pyran copolymer, or thioglycollate yielded less PGI2. Reductions ranged from 73% for C. parvum to 32% for levamisole. A statistically significant inverse correlation existed between the level of macrophage "activation" and ability of cellular homogenates to produce prostacyclin. The results suggest that prostacyclin may be involved in modulation of nonspecific resistance.     

Prostacyclin biosynthesis in activated, stimulated and normal mouse peritoneal cell populations

Nonspecific resistance to infectious and neoplastic disease can be enhanced by administration of “immunomodulators”. The levels of enhancement can be monitored by following function of cells of the lympho-reticuloendothelial system. To gain a better understanding of the physiological and biochemical nature of this enhancement, the metabolism of prostaglandin endoperoxide PGH₂ was followed in mouse peritoneal cells (PCs). Homogenates of PCs from normal, unstimulated mice yielded primarily prostacyclin (PGI₂) when incubated with PGH₂. Homogenates of PCs from mice injected with the immunomodulators . , levamisole HCl, pyran copolymer, or thioglycollate yielded less PGI₂. Reductions ranged from 73% for . to 32% for levamisole. A statistically significant inverse correlation existed between the level of macrophage “activation” and ability of cellular homogenates to produce prostacyclin. The results suggest that prostacyclin may be involved in modulation of nonspecific resistance.     

The keratin polypeptide patterns in heterotypically recombined epithelia of skin and mucosa of adult mouse

Epithelial-mesenchymal interactions were investigated considering both morphologic criteria and keratin polypeptide expression in homotypic and heterotypic recombinants of adult mouse skin and oral mucosa. Two series of cross-recombinants of epithelia with different morphology and keratin patterns were chosen: (a) footpad epidermis/ear dermis and ear epidermis/footpad dermis; (b) palate epithelium/cheek connective tissue and cheek epithelium/palate connective tissue. Homotypic and heterotypic recombinants were prepared after EDTA-separation of the original tissues and then grown on syngeneic mice in subcutaneously prepared protected graft chambers. EDTA-separation is especially suited to completely separate the epidermal-dermal union, and the transplantation procedure used strictly prevents contamination with host epithelium. Five weeks after implantation keratins were analyzed by one and two-dimensional gel electrophoresis and peptide mapping. In both series, homotypic recombination of the tissues did not alter the original morphology and keratin polypeptide composition of the individual epithelial components. Ear epidermis displayed no significant changes in structure or keratin pattern in heterotypic recombinants. Recombined with ear dermis, footpad epidermis showed acquisition of some morphologic features typical for ear epidermis and slight changes in keratin composition which were, however, difficult to interpret due to the normal similarities of footpad keratin with that of ear. In contrast, the heterorecombinants of the palate/cheek series exhibited considerable alterations in their keratin patterns. Either epithelium showed suppression of distinct keratin subunits and de novo expression of subunits characteristic of the epithelium normally associated with the connective tissue component. The keratin patterns of both matches closely resembled each other and represented patterns intermediate between the normal patterns. This partial, however, significant modulation in the expression of differentiation markers was paralleled by similarly directed changes in the architecture of the heterotransplanted tissues, thus indicating that both morphogenesis and cytodifferentiation of certain adult epithelia can be influenced by extrinsic mesenchymal factors.     

Expression of basal cell keratin 15 and keratin 19 in oral squamous neoplasms represents diverse pathophysiologies

Human epithelium contains keratin, which is expressed during differentiation. Depending on the target cell type, different types of keratin are expressed, and their alterations seem to represent changes in cell properties. The basal cells of oral epithelium express keratin 5 (K5), K14, K15 and K19, but their alterations in tumors are unclear. To address this issue and to seek possible diagnostic application, we examined the expression of these keratins in oral squamous cell carcinoma (OSCC) and squamous intraepithelial neoplasm (SIN). cDNA microarray analysis of 43 OSCC revealed slight upregulation of KRT14, downregulation of KRT15 and KRT19, and unaltered KRT5 expression. There were great variations in KRT15 and KRT19 expression across each cancer. Well-differentiated OSCC tended to express more KRT15 and less KRT19 compared to moderately- or poorly-differentiated OSCC. KRT15 was positively correlated with differentiation-related keratin, KRT13. These observations were further investigated by immunohistochemical examination. K5 and K14 were ubiquitously expressed in all 50 OSCC and 50 SIN examined. K15 and K19 were generally downregulated, but were considerably retained in about half of the cases and showed diverse expression patterns. K15-positive cancers tended to show a well-differentiated phenotype, and K19-positive cancers tended to show more invasive tumor fronts. Most K19-positive cancers appeared to develop with little associating SIN. K19 was consistently downregulated in SIN, while K15 was downregulated mainly in high grade SIN. In summary, K15 and K19, unlike K5 or K14, are expressed variably in both SIN and OSCC, which reflects the differences in their pathogenesis and biological behaviors, suggesting their prospective applications as markers for subclassifying OSCC and SIN.     

Local variations in the staining patterns of keratin antigens and blood group antigens in normal rodent oral epithelia

Summary All rodent oral epithelia are orthokeratinized. However, morphological, immunohistochemical and biochemical studies have shown that regional differences exist. In the present study, intraregional variations in differentiation patterns of rat oral epithelia are demonstrated using monoclonal anti-keratin antibodies AE1 and AE2 and antibodies to blood group antigens B and H. Well-defined areas of rat buccal and hard palate epithelium differed from the general staining patterns of these epithelia. These areas were associated with a papillary surface contour. These local variations were not found in the strain of mice examined. The results suggest that physiologically different vertical compartments of keratinocytes exist within one and the same region of rat oral mucosa, a phenomenon previously recognized in detail only in the epithelium of dorsal tongue. The papillary structures may have some functional significance related to the processing of food similar to that suggested for lingual filiform papillae.     

Polarized and functional epithelia can form after the targeted inactivation of both mouse keratin 8 alleles

We have tested the requirement of keratin intermediate filaments for the formation and function of a simple epithelium. We disrupted both alleles of the mouse keratin 8 (mK8) gene in embryonic stem cells, and subsequently analyzed the phenotype in developing embryoid bodies in suspension culture. After the inactivation of the mouse keratin 8 (mK8) gene by a targeted insertion, mK8 protein synthesis was undetectable. In the absence of mK8 its complementary partners mK18 and mK19 were unable to form filaments within differentiated cells. Surprisingly, these ES cells differentiate to both simple and cystic embryoid bodies with apparently normal epithelia. Ultrastructural analysis shows an apparently normal epithelium with microvilli on the apical membrane, tight junctions and desmosomes on the lateral membrane, and an underlying basal membrane. No significant differences in the synthesis or secretion of alpha 1-fetoprotein and laminin were observed between the mK8- or wild-type embryoid bodies. Our data show that mK8 is not required for simple epithelium formation of extraembryonic endoderm.     

Effect of hormones on primary cell cultures from pregnancy-dependent mouse mammary tumors.

No hormone combination was successful in maintaining long term primary cultures of pregnancy-dependent mammary tumors. Insulin provided the most consistent dose dependent stimulatory effect on short term proliferation as measured by 3H-TdR incorporation into DNA. Insulin in combination with corticosterone and prolactin produced the greatest stimulatory effect in most tumors. Both insulin at 5 microgram/ml and insulin, prolactin and hydrocortisone induced a partially synchronous wave of DNA synthesis in restimulated cultures. The time course of the wave of DNA synthesis was different for different hormone treatments. Insulin as 5 microgram/ml caused an earlier wave of DNA synthesis than insulin, prolactin plus corticosterone.     

Protein phosphorylation substrates in normal and neoplastic squamous epithelia of the human upper aero-digestive tract

Protein phosphorylation was studied in crude and in protein kinase C (Pk-C)-enriched preparations from squamous cell carcinomas and normal mucosa of the human upper aero-digestive tract. In crude soluble preparations from neoplastic mucosa we found a 5-fold higher basal endogenous phosphorylation when compared to normal mucosa. In particulate fractions the increase was 3-fold. SDS-PAGE and autoradiography of phosphorylated proteins in crude soluble tumor extracts showed bands corresponding to proteins with apparent molecular weights of 18, 37, 40-42, 52, 60, 62 and 90 kDa. In normal mucosa the phosphorylation of these proteins was very low or absent, except for the proteins with molecular weights of 40-42 and 52-55 kDa. Addition of Ca2+ or Ca2+/phospholipids to the reaction mixture caused phosphorylation of additional proteins with apparent molecular weight of 45-50 kDa in soluble preparations of tumors. Cyclic AMP or cGMP had no significant effect on the phosphorylation of endogenous proteins. In the partially purified, Pk-C-enriched fractions the phosphorylation in the presence of Ca2+/phospholipids was distinctly higher in tumors when compared to the phosphorylation observed in normal mucosa, and some phosphorylation substrates were detected only in tumor tissue. In order to find out whether the elevated basal phosphorylation was due to an endogenous activation of protein kinases, different inhibitors of serine/threonine protein kinases were tested. These inhibitors included: heat-stable cyclic AMP-dependent protein kinase (Pk-A) inhibitor, Pk-A inhibitor peptide (Wiptide), heparin and the Pk-C inhibitors peptide 19-36 and H-7. None of these inhibitors had any significant effect on the basal phosphorylation. In conclusion, our results show the existence of endogenous phosphorylation substrates in human squamous cell carcinomas from the upper aerodigestive tract, and indicates that there is a significantly higher basal and Pk-C specific phosphorylation of endogenous substrates in tumors compared to normal mucosa. This may be of importance for the transformation and altered growth regulation in epithelial tumors.     

Keratin polypeptide distribution in benign and malignant breast tumors: subdivision of ductal carcinomas using monoclonal antibodies

Monoclonal antibodies which recognize one or only a few keratin polypeptides have been used to study the distribution of different keratins in benign and malignant breast lesions by immunocytochemical methods. Seven monoclonal antibodies which recognized either different keratin polypeptides by immunoblotting techniques, or identified different epithelial cell types in complex tissues were used. In two mastopathies and three fibroadenomas the antibody lu5 stained luminal cells as well as myoepithelial cells. In contrast the antibodies CK7, Troma 1, CK2 and KA4 labeled only luminal cells, whereas antibody CKB1 decorated only myoepithelial cells. All 15 ductal carcinomas showed a uniform staining of tumor cells with the antibodies Troma 1, CK2, KA4 and lu5. The antibody CK7 also stained all ductal carcinomas, but in two specimens the staining was heterogeneous. The antibody CKB1 decorated only the pre-existing myoepithelial cells in 11 of 12 ductal carcinomas but in the remaining specimen the tumor cells were also strongly positive. Tumor cells in lobular carcinomas were labeled by antibodies CK7, Troma 1, CK2, KA4, bu not by CKB1. The antibody CKS1 showed no staining of any of the benign and malignant breast lesions.     

Expression of cyclo-oxygenase-2 in naturally occurring squamous cell carcinomas in dogs.

Squamous cell carcinoma is one of the most common cancers in humans and is also a frequently diagnosed neoplasm in dogs. Induction of cyclo-oxygenase-2 (COX-2), a key rate-limiting enzyme in prostaglandin biosynthesis, has been implicated in the oncogenesis of various cancers in humans, including squamous cell carcinomas. However, expression of COX-2 has not been reported in spontaneous squamous cell carcinomas of non-human species. Canine squamous cell carcinomas share several similarities with the human disease. Therefore, the objective of this study was to determine whether COX isoenzymes were expressed in naturally occurring cases of squamous cell carcinomas in dogs. Canine normal skin (n=4) and squamous cell carcinomas (n=40) were studied by immunohistochemistry and immunoblotting analysis using polyclonal antibodies selective for COX-1 or COX-2. COX-2 was strongly expressed by neoplastic keratinocytes in all cases of squamous cell carcinomas, whereas no COX-2 was detected in normal skin and in the non-neoplastic skin and oral mucosa included in the tumor tissue samples (p<0.01). Immunoblotting analysis confirmed the restricted expression of COX-2 (72,000--74,000 molecular weight doublet) in squamous cell carcinomas only. In contrast, faint COX-1 staining was found in normal skin and in squamous cell carcinomas. This study demonstrates for the first time that COX-2 is induced in canine squamous cell carcinomas, and provides a new model to investigate the role and regulation of COX-2 gene expression in naturally occurring squamous cell carcinomas. (J Histochem Cytochem 49:867-875, 2001)     

Tight junction-related structures in the absence of a lumen: occludin, claudins and tight junction plaque proteins in densely packed cell formations of stratified epithelia and squamous cell carcinomas

Tight junctions (TJs), hallmark structures of one-layered epithelia and of endothelia, are of central biological importance as intramembranous "fences" and as hydrophobic "barriers" between lumina represented by liquid- or gas-filled spaces on the one hand and the mesenchymal space on the other. They have long been thought to be absent from stratified epithelia. Recently, however, constitutive TJ proteins and TJ-related structures have also been identified in squamous stratified epithelia, including the epidermis, where they occur in special positions, most prominently in the uppermost living epidermal cell layer, the stratum granulosum. Much to our surprise, however, we have now also discovered several major TJ proteins (claudins 1 and 4, occludin, cingulin, symplekin, protein ZO-1) and TJ-related structures in specific positions of formations of epithelium-derived tissues that lack any lumen and do not border on luminal or body surfaces. Using immunohistochemistry and electron microscopy we have localized TJ proteins and structures in peripheral cells of the Hassall's corpuscles of human and bovine thymi as well as in specific central formations of tumor nests in squamous cell carcinomas, including the so-called "horn pearls". Such structures have even been found in carcinoma metastases. In carcinomas, they often seem to separate certain tumor regions from others or from stroma. The structural significance and the possible functional relevance of the locally restricted synthesis of TJ proteins and of the formations of TJ-related structures are discussed. It is proposed to include the determination of the presence or absence of such proteins and structures in the diagnostic program of tumor pathology.     

Solid-state NMR studies of the dynamics and structure of mouse keratin intermediate filaments

The molecular dynamics and structural organization of mouse epidermal keratin intermediate filaments (IF) have been studied via solid-state nuclear magnetic resonance (NMR) experiments performed on IF labeled both in vivo and in vitro with isotopically enriched amino acids. As a probe of the organization of the peripheral glycine-rich end domains of the IF, carbon-13 NMR experiments have been performed on subfilamentous forms (prekeratin) and on IF reassembled in vitro that had been labeled with either [1-13C]glycine or [2-13C]glycine, as more than 90% of the glycines of the keratins are located in the end domains. Although cross-labeling to seryl residues was observed, the proportion of serine located in the end domains is nearly the same as that for glycine. Measurements of carbon relaxation times, nuclear Overhauser enhancements, and signal intensities show that the motions of the peptide backbone in the end domains are effectively isotropic, with average correlation times distributed over the range of 0.2-20 ns. These results indicate that the end domains of IF are remarkably flexible and have little or no structural order. To probe the structural organization of the coiled-coil rod domains of the IF, separate samples of native keratin IF, raised in primary tissue culture, were labeled with L-[1-13C]leucine, L-[2H10]leucine, or L-[2,3,3-2H3]leucine, as greater than 90% of the leucyl residues of the keratin IF types studied are located in the coiled coils which form the central core of IF.(ABSTRACT TRUNCATED AT 250 WORDS)     

Allelic imbalance in primary breast carcinomas and metastatic tumors of the axillary lymph nodes

Axillary lymph node status is the most important prognostic factor in predicting disease outcome in women with breast cancer. A number of chromosomal aberrations in primary breast tumors have been correlated with lymph node status and clinical outcome, but chromosomal changes particular to metastatic lymph node tumors have not been well studied. DNA samples isolated from laser-microdissected primary breast and metastatic axillary lymph node tumors from 25 women with invasive breast cancer were amplified using 52 microsatellite markers defining 26 chromosomal regions commonly deleted in breast cancer. Levels and patterns of allelic imbalance (AI) within and between breast and lymph node tumors were assessed to identify chromosomal alterations unique to primary or metastatic tumors and to examine the timing of metastatic potential. The overall frequency of AI in primary breast tumors (0.24) was significantly greater (P < 0.001) than that in lymph node tumors (0.10), and congruent AI events were observed for < 20% of informative markers. AI at chromosomes 11q23.3 and 17p13.3 occurred significantly more frequently (P < 0.05) in primary breast tumors alone; no chromosomal regions showed a significantly higher AI frequency in lymph nodes. Higher rates of AI in primary versus metastatic lymph node tumors suggest that acquisition of metastatic potential may be an early event in carcinogenesis, occurring before significant levels of AI accumulate in the primary tumor. In addition, patterns of AI were highly discordant between tumor types, suggesting that additional genetic alterations accumulated independently in the two cell populations.     

Promoter methylation in head and neck squamous cell carcinoma cell lines is significantly different than methylation in primary tumors and xenografts

Studies designed to identify novel methylation events related to cancer often employ cancer cell lines in the discovery phase of the experiments and have a relatively low rate of discovery of cancer-related methylation events. An alternative algorithm for discovery of novel methylation in cancer uses primary tumor-derived xenografts instead of cell lines as the primary source of nucleic acid for evaluation. We evaluated DNA extracted from primary head and neck squamous cell carcinomas (HNSCC), xenografts grown from these primary tumors in nude mice, HNSCC-derived cell lines, normal oral mucosal samples, and minimally transformed oral keratinocyte-derived cell lines using Illumina Infinum Humanmethylation 27 genome-wide methylation microarrays. We found >2,200 statistically significant methylation differences between cancer cell lines and primary tumors and when comparing normal oral mucosa to keratinocyte cell lines. We found no statistically significant promoter methylation differences between primary tumor xenografts and primary tumors. This study demonstrates that tumor-derived xenografts are highly accurate representations of promoter methylation in primary tumors and that cancer derived cell lines have significant drawbacks for discovery of promoter methylation alterations in primary tumors. These findings also support use of primary tumor xenografts for the study of methylation in cancer, drug discovery, and the development of personalized cancer treatments.