A truncated SV40 large T antigen lacking the p53 binding domain overcomes p53-induced growth arrest and immortalizes primary mesencephalic cells

As an alternative to primary fetal tissue, immortalized central nervous system (CNS)-derived cell lines are useful for in vitro CNS model systems and for gene manipulation with potential clinical use in neural transplantation. However, obtaining immortalized cells with a desired phenotype is unpredictable, because the molecular mechanisms of growth and differentiation of CNS cells are poorly understood. The SV40 large T antigen is commonly used to immortalize mammalian cells, but it interferes with multiple cell-cycle components, including p53, p300, and retinoblastoma protein, and usually produces cells with undifferentiated phenotypes. In order to increase the phenotypic repertoire of immortalized CNS cells and to address the molecular mechanisms underlying immortalization and differentiation, we constructed an expression vector containing a truncated SV40 large T gene that encodes only the amino-terminal 155 amino acids (T155), which lacks the p53-binding domain. Constructs were first transfected into a p53-temperature-sensitive cell line, T64-7B. Colonies expressing T155 proliferated at the growth-restrictive temperature. T155 was then transfected into primary cultures from embryonic day-14 rat mesencephalon. Two clonal cell lines were derived, AF-5 and AC-10, which co-expressed T155 and mature neuronal and astrocytic markers. Thus, the amino-terminal portion of SV40 large T is sufficient to: (1) overcome p53-mediated growth arrest despite the absence of a p53-binding region, and (2) immortalize primary CNS cells expressing mature markers while actively dividing. T155 and T155-transfectants may be useful for further studies of cell-cycle mechanisms and phenotyic expression in CNS cells or for further gene manipulation to produce cells with specific properties.     

Establishment of murine cell lines by constitutive and conditional immortalization

Mouse cell lines were immortalized by introduction of specific immortalizing genes. Embryonic and adult animals and an embryonal stem cell line were used as a source of primary cells. The immortalizing genes were either introduced by DNA transfection or by ecotropic retrovirus transduction. Fibroblasts were obtained by expression of SV40 virus large T antigen (TAg). The properties of the resulting fibroblast cell lines were reproducible, independent of the donor mouse strains employed and the cells showed no transformed properties in vitro and did not form tumors in vivo. Endothelial cell lines were generated by Polyoma virus middle T antigen expression in primary embryonal cells. These cell lines consistently expressed relevant endothelial cell surface markers. Since the expression of the immortalizing genes was expected to strongly influence the cellular characteristics fibroblastoid cells were reversibly immortalized by using a vector that allows conditional expression of the TAg. Under inducing conditions, these cells exhibited properties that were highly similar to the properties of constitutively immortalized cells. In the absence of TAg expression, cell proliferation stops. Cell growth is resumed when TAg expression is restored. Gene expression profiling indicates that TAg influences the expression levels of more than 1000 genes that are involved in diverse cellular processes. The data show that conditionally immortalized cell lines have several advantageous properties over constitutively immortalized cells.     

Establishment of immortal swine kidney epithelial cells

Using normal swine kidney epithelial (SKE) cells that were shown to be senescent at passages 12 to 14, we have established one lifespan-extended cell line and two lifespan-extended cell lines by exogenous introduction of the human catalytic subunit of telomerase (hTERT) and simian virus 40 large T-antigen (SV40LT), all of which maintain epithelial morphology and express cytokeratin, a marker of epithelial cells. SV40LT- and hTERT-transduced immortal cell lines appeared to be smaller and exhibited more uniform morphology relative to primary and spontaneously immortalized SKE cells. We determined the in vitro lifespan of primary SKE cells using a standard 3T6 protocol. There were two steps of the proliferation barrier at 12 and 20, in which a majority of primary SKE cells appeared enlarged, flattened, vacuolated, and ss-galactosidase-positive, all phenotypical characteristics of senescent cells. Lifespan-extended SKE cells were eventually established from most of the cellular foci, which is indicative of spontaneous cellular conversion at passage 23. Beyond passage 25, the rate of population doubling of the established cells gradually increased. At passage 30, immortal cell lines grew faster than primary counterpart cells in 10% FBS-DMEM culture conditions, and only SV40LT-transduced immortal cells grew faster than primary and other SKE immortal cells in 0.5% FBS-DMEM. These lifespan-extended SKE cell lines failed to grow in an anchorage-independent manner in soft-agar dishes. Hence, three immortalized swine kidney epithelial cells that are not transformed would be valuable biological tools for virus propagation and basic kidney epithelial cell research.     

Antigenic and functional characterization of a rat central nervous system-derived cell line immortalized by a retroviral vector

We have immortalized rat central nervous system (CNS) cells of primary cultures of rat optic nerve with murine leukemia virus psi-2,SV-40-6, which is defective in assembly and contains the SV-40 large T antigen and neomycin resistance genes, to produce a cell line that we named A7. After drug selection, greater than 90% of the growing cells expressed nuclear SV-40 large T cells and a fraction of these contained the astrocyte-specific marker, glial fibrillary acidic protein. The majority of these cells also expressed surface marker A4 (specific for neural tube derivatives), Ran 2, p185 (the 185-kD phosphoprotein product of the neu oncogene), and fibronectin, but did not express the astrocyte enzymes glutamine synthetase and monoamine oxidase B. Surface markers characteristic of glial progenitors (A2B5) and oligodendrocytes (galactocerebroside) were not detected. After two rounds of cell cloning, subclone A7.6-3 expressed Ran 2, fibronectin, and the neural cell adhesion molecule (N-CAM) but not glial fibrillary acidic protein and A4. The A7 cell line and subclones also displayed certain functions of type 1 astrocytes: the conditioned medium of these cells had a potent mitogenic activity for glial progenitor cells which could be neutralized by anti-platelet-derived growth factor antibodies and monolayers of these cells supported the growth of embryonic hypothalamic neurons. We conclude that a retrovirus containing SV-40 large T antigen can immortalize rat CNS cells and that such immortalized glial cells retain at least two important functions of type 1 astrocytes: the ability to secrete platelet-derived growth factor and to support the growth of embryonic CNS neurons. Moreover, such stable immortalized clonal cell lines can be used to study gene regulation in glial cells.     

Inhibition of human papillomavirus type 16 E7 phosphorylation by the S100 MRP-8/14 protein complex

The human papillomavirus type 16 (HPV16) E7 is a major viral oncoprotein that is phosphorylated by casein kinase II (CKII). Two S100 family calcium-binding proteins, macrophage inhibitory-related factor protein 8 (MRP-8) and MRP-14, form a protein complex, MRP-8/14, that inactivates CKII. The MRP-8/14 protein complex may inhibit CKII-mediated E7 phosphorylation and therefore may alter its interaction with cellular ligands and reduce E7 oncogenic activity. We examined the inhibitory effect of the MRP-8/14 complex on CKII activity and HPV16 E7 phosphorylation. We have shown that CKII activity and HPV16 E7 phosphorylation were inhibited by uptake of exogenous MRP-8/14 and activation of endogenous MRP-8/14. MRP-8/14-mediated inhibition of E7 phosphorylation occurred at the G1 phase of the cell cycle. Analysis of MRP expression in primary keratinocytes and in HPV16- and 18-transformed cervical and foreskin epithelial cell lines showed that expression of MRP-8, MRP-14, and the MRP-8/14 complex was detected only in primary untransformed keratinocytes and not in the HPV-infected immortalized epithelial cells. CKII activity in HPV-immortalized keratinocytes was approximately fourfold higher than in HPV-negative primary keratinocytes. Treatment of HPV-positive immortalized epithelial cells with exogenous MRP-8/14 resulted in E7 hypophosphorylation and complete inhibition of cell growth within 2 weeks, compared with HPV-negative primary and immortalized HPV-negative cervical epithelial cells, which showed 25 and 40% growth inhibition, respectively. Together these results suggests that the MRP-8/14 protein complex in HPV-infected epithelial cells may play an important role in regulation of CKII-mediated E7 phosphorylation and inhibition of its oncogenic activity.     

SV40 immortalizes myogenic cells: DNA synthesis and mitosis in differentiating myotubes

Primary skeletal muscle myoblasts have a limited proliferative capacity in cell culture and cease to proliferate after several passages. We examined the effects of several oncogenes on the immortalization and differentiation of primary cultures of rat skeletal muscle myoblasts. Retroviruses containing a SV40 large T antigen (LT) gene very efficiently immortalize myogenic cells. The immortalized cell lines retain a very high differentiation capacity and form, in the appropriate culture conditions, a very dense network of muscle fibers. As in primary culture, cell fusion is associated with the synthesis of large amounts of muscle-specific proteins. However, unlike normal myoblasts (and previously established myogenic cell lines), nuclei in the multinucleated fibers of SV40-immortalized cells synthesize DNA and enter mitosis. Thus, withdrawal from DNA synthesis is not obligatory for cell fusion and biochemical differentiation. Using a retrovirus coding for a temperature-sensitive SV40 LT, myogenic cell lines were produced in which the SV40 LT could be inactivated by a shift from 33 degrees C to 39 degrees C. The inactivation of LT induced massive cell fusion and synthesis of muscle proteins. The nuclei in those fibers did not synthesize DNA, nor did they undergo mitosis. This approach enabled the reproducible establishment of myogenic cell lines from very small populations of myoblasts or single primary myogenic clones. Activated p53 also readily immortalized cells in primary muscle cultures, however the cells of eight out of the nine cell lines isolated had a fibroblastic morphology and could not be induced to form multinucleated fibers.     

Immortalization of germ cells and somatic testicular cells using the SV40 large T antigen

We report the immortalization, using the SV40 large T antigen, of all the cell types contributing to a developing seminiferous tubule in the mouse testis. Sixteen peritubular, 22 Leydig, 8 Sertoli, and 1 germ cell line have been established and cultured successfully for 90 generations in a period of 2.5 years. Immortalized peritubular cells were identified by their spindle-like appearance, their high expression of alkaline phosphatase, and their expression of the intermediary filament desmin. They also produce high amounts of collagen. Immortalized Leydig cells are easily identifiable by the accumulation of lipid droplets in their cytoplasm and the production of the enzyme 3-beta-hydroxysteroid dehydrogenase. Some Leydig cell lines also express LH receptors. The immortalized Sertoli cells are able to adopt their typical in vivo columnar appearance when cultured at high density. They exhibit a typical indented nucleus and cytoplasmic phagosomes. Some Sertoli cell lines also express FSH receptors. A germ cell line (GC-1spg) was established that corresponds to a stage between spermatogonia type B and primary spermatocyte, based on its characteristics in phase contrast and electron microscopy. This cell line expresses the testicular cytochrome ct and lactate dehydrogenase-C4 isozyme. These four immortalized cell types, when plated together, are able to reaggregate and form structures resembling two-dimensional spermatogenic tubules in vitro. When only the immortalized somatic cells are cocultured, the peritubular and Sertoli cells form cord-like structures in the presence of Leydig cells. Fresh pachytene spermatocytes cocultured with the immortalized somatic cells integrate within the cords and are able to survive for at least 7 days. The ability to perform coculture experiments with immortalized testicular cell lines represents an important advancement in our ability to study the nature of cell-cell and cell-matrix interactions during spermatogenesis and testis morphogenesis.     

Conditional immortalization of normal and dysgenic mouse muscle cells by the SV40 large T antigen under the vimentin promoter control.

We have created new mouse muscle cell lines of an immortalized type, expressing normal differentiation at the myotube stage: sarcomeric organization, functional excitation-contraction coupling, and triadic differentiation. The DNA immortalizing recombinant utilizes a deletion mutant of the regulatory region of the human vimentin promoter controlling the expression of a SV40 thermosensitive large T antigen, in which the small t sequence has been deleted. Skeletal mouse replicative myoblasts synthesized predominantly vimentin. After myoblast fusion the vimentin gene is strongly repressed in multinucleated syncytia. Furthermore, the normal activity of the vimentin promoter in myoblasts is increased in the large T antigen-expressing cells. We observed that continuous and rapid division of myoblasts occurs at permissive temperature, suggesting that immortalization is achieved even though the small t antigen is absent. When fusion is induced by changing media conditions, large T antigen expression is totally repressed by the vimentin promoter. When the temperature is elevated to 39 degrees C, the preexisting large T antigen is inactivated. The resulting myotubes from normal mouse differentiate totally normally as indicated by their morphology, ultrastructure, and electrophysiological properties. Mutant (muscular dysgenesis) immortalized cells express the same properties as mutant primary counterparts with no contraction, no slow Ca2+ current, and no triadic differentiation. These immortalized cell lines are potentially very useful for further pharmacology, transplantation, and cell biology studies. The vimentin promoter control of immortalizing recombinant DNA can be used for any mammalian normal and mutant muscle cell lines.     

Establishment and characterization of endothelial cell lines from the aorta of miniature pig for the study of xenotransplantation

Pig endothelial cells are the first cells to interact with human immune components after organ xenotransplantation, which is a procedure currently considered to be the best treatment option for end-stage organ failure. It is, therefore, essential to study the mechanisms of molecular interaction between pig endothelial cells and human immune components, in order to overcome xenograft rejection. The aim of this study was to establish immortalized pig aortic endothelial cell lines, in order to facilitate future in vitro studies of human anti-pig immune responses. Endothelial cell lines were established following the transfection of primary endothelial cells isolated from the aortas of the Minnesota miniature pig with plasmid pRNS-1 carrying genes for neomycin resistance and the SV40 large T antigen. The immortalized cell lines showed a relatively rapid doubling time (17.6h) and the endothelial cell phenotype, as indicated by the formation of typical cobblestone monolayers and by the constitutive expression of PECAM-1 and the von Willebrand factor. Flow cytometric analysis demonstrated the constitutive expression of SLA class I and CD86, whereas the expression of E-selectin and SLA class II was only induced after stimulation with human TNF-alpha and pig IFN-gamma, respectively. On the other hand, no CD80 expression was detected in the primary cells or cell lines in the presence or absence of either human TNF-alpha or pig IFN-gamma. A vigorous human T cell proliferation against these cell lines was observed in the mixed lymphocyte-endothelial cell culture. These results suggest that pig endothelial cells, immortalized by the introduction of SV40 T, retain their original characteristics, except for the acquired property of immortalization, and that they may be useful for future in vitro studies of xenogeneic human anti-pig immune responses.     

Human papillomavirus E6 proteins bind p53 in vivo and abrogate p53-mediated repression of transcription.

The transforming proteins of DNA tumor viruses SV40, adenovirus and human papillomaviruses (HPV) bind the retinoblastoma and p53 cell cycle regulatory proteins. While the binding of SV40 large T antigen and the adenovirus E1B 55 kDa protein results in the stabilization of the p53 protein, the binding of HPV16 and 18 E6 results in enhanced degradation in vitro. To explore the effect of viral proteins on p53 stability in vivo, we have examined cell lines immortalized in tissue culture by HPV18 E6 and E7 or SV40 large T antigen, as well as cell lines derived from cervical neoplasias. The half-life of the p53 protein in non-transformed human foreskin keratinocytes in culture was found to be approximately 3 h while in cell lines immortalized by E6 and E7, p53 protein half-lives ranged from 2.8 h to less than 1 h. Since equivalent levels of E6 were found in these cells, the range in p53 levels observed was not a result of variability in amounts of E6. In keratinocyte lines immortalized by E7 alone, the p53 half-life was found to be similar to that in non-transformed cells; however, it decreased to approximately 1 h following supertransfection of an E6 gene. These observations are consistent with an interaction of E6 and p53 in vivo resulting in reductions in the stability of p53 ranging between 2- and 4-fold. We also observed that the expression of various TATA containing promoters was repressed in transient assays by co-transfection with plasmids expressing the wild-type p53 gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth of immortal simian virus 40 tsA-transformed human fibroblasts is temperature dependent.

Simian virus 40 (SV40)-mediated transformation of human fibroblasts offers an experimental system for studying both carcinogenesis and cellular aging, since such transformants show the typical features of altered cellular growth but still have a limited life span in culture and undergo senescence. We have previously demonstrated (D. S. Neufeld, S. Ripley, A. Henderson, and H. L. Ozer, Mol. Cell. Biol. 7:2794-2802, 1987) that transformants generated with origin-defective mutants of SV40 show an increased frequency of overcoming senescence and becoming immortal. To clarify further the role of large T antigen, we have generated immortalized transformants by using origin-defective mutants of SV40 encoding a heat-labile large T antigen (tsA58 transformants). At a temperature permissive for large-T-antigen function (35 degrees C), the cell line AR5 had properties resembling those of cell lines transformed with wild-type SV40. However, the AR5 cells were unable to proliferate or form colonies at temperatures restrictive for large-T-antigen function (39 degrees C), demonstrating a continuous need for large T antigen even in immortalized human fibroblasts. Such immortal temperature-dependent transformants should be useful cell lines for the identification of other cellular or viral gene products that induce cell proliferation in human cells.     

东方田鼠胚胎成纤维细胞永生化细胞系的建立

目的建立东方田鼠胚胎成纤维永生化细胞系,为全面研究东方田鼠抗日本血吸虫机制以及开展不同动物成纤维细胞间比较研究奠定基础和提供细胞实验材料。方法运用脂质体介导的基因转染法将pSV3neo质粒导入第3代东方田鼠胚胎成纤维细胞,经G418筛选抗性克隆并扩大培养,建立永生化细胞系;用PCR检测细胞株中SV40T基因的整合,RT-PCR鉴定SV40T基因在转染细胞中的表达;绘制东方田鼠胚胎成纤维永生化细胞生长曲线。结果阳性细胞克隆已扩大培养并稳定传代50代,经鉴定SV40T抗原已整合到东方田鼠胚胎成纤维细胞中且稳定表达。结论成功建立东方田鼠胚胎成纤维永生化细胞系。     

Immortalized Human Schwann Cell Lines Derived From Tumors of Schwannomatosis Patients

Schwannomatosis, a rare form of neurofibromatosis, is characterized predominantly by multiple, often painful, schwannomas throughout the peripheral nervous system. The current standard of care for schwannomatosis is surgical resection. A major obstacle to schwannomatosis research is the lack of robust tumor cell lines. There is a great need for mechanistic and drug discovery studies of schwannomatosis, yet appropriate tools are not currently available. Schwannomatosis tumors are difficult to grow in culture as they survive only a few passages before senescence. Our lab has extensive experience in establishing primary and immortalized human Schwann cell cultures from normal tissue that retain their phenotypes after immortalization. Therefore we took on the challenge of creating immortalized human Schwann cell lines derived from tumors from schwannomatosis patients. We have established and fully characterized 2 schwannomatosis cell lines from 2 separate patients using SV40 virus large T antigen. One patient reported pain and the other did not. The schwannomatosis cell lines were stained with S100B antibodies to confirm Schwann cell identity. The schwannomatosis cells also expressed the Schwann cell markers, p75NTR, S100B, and NGF after multiple passages. Cell morphology was retained following multiple passaging and freeze/ thaw cycles. Gene expression microarray analysis was used to compare the cell lines with their respective parent tumors. No differences in key genes were detected, with the exception that several cell cycle regulators were upregulated in the schwannomatosis cell lines when compared to their parent tumors. This upregulation was apparently a product of cell culturing, as the schwannomatosis cells exhibited the same expression pattern of cell cycle regulatory genes as normal primary human Schwann cells. Cell growth was also similar between normal primary and immortalized tumor cells in culture. Accurate cell lines derived directly from human tumors will serve as invaluable tools for advancing schwannomatosis research, including drug screening.     

Two classes of transformation-deficient, immortalization-positive simian virus 40 mutants constructed by making three-base insertions in the T antigen gene

We constructed two mutants of simian virus 40 (SV40) by introducing a three-base duplication at AvaII cutting sites within the large T antigen coding region, and we examined these mutants for their abilities to replicate in monkey GC7 cells, to transform rat cell line 3Y1 cells, and to transform and immortalize primary cells from newborn rats. Neither of the mutants could replicate in GC7 cells. One mutant with the duplication at 0.335 SV40 map units (m.u.) (inA942) could transform 3Y1 cells, but the other mutant with the duplication at 0.636 m.u. (inA941) could not. The two mutants could not transform primary rat cells but retained immortalization activity. The results suggest that transformation of primary cells by SV40 requires at least two distinct activities of the large T antigen, one of which can be replaced by a cellular function(s) expressed in immortalized 3Y1 cells.     

Rat Sertoli cells express epithelial but also mesenchymal genes after immortalization with SV40

A new immortal Sertoli cell line from pubertal rat testis was established and characterized. We have generated the clonal line SCIT-C8 expressing established markers for Sertoli cells (SC) like transferrin, clusterin and steel factor/stem cell factor (SCF). Additionally, the immortalized cells express afadin, a protein which is a member of tight and adherens junctions, therefore the cells may be useful for studies of the blood-testis barrier (BTB) in vitro. In contrast to primary SC, the immortalized cells lost expression of androgen receptor and responsiveness to androgens and follicle-stimulating hormone. Surprisingly, we found mRNA expression and protein secretion of the mesenchymal markers, fibronectin and entactin-1, which we also observed for the immortalized SC lines, ASC-17D and 93RS2. In comparison to primary SC, the immortalized cells demonstrated enhanced adhesion in vitro. This correlated with the expression of entactin-1 because adhesion was strongly reduced by antibody perturbation experiments. Additionally, we found the alternatively spliced and primarily muscle cell-specific long variant of TGF-beta2 not only in peritubular cells (PC), but also in the primary and immortalized SC. Furthermore, all immortalized cell lines secreted higher amounts of TGF-beta2 than primary SC. In conclusion, the immortalized SC lines from different developmental stages showed a similar pattern of epithelial and mesenchymal markers.     

Establishment of two immortalized nasopharyngeal epithelial cell lines using SV40 large T and HPV16E6/E7 viral oncogenes

Nasopharyngeal carcinoma (NPC) is a common cancer in Southeast Asia, especially in southern China. One of the most striking features of this disease is its close relationship with Epstein-Barr Virus (EBV). However, to date there is no direct study on the mechanisms involved in the role of EBV in the tumorigenesis of NPC, largely due to lack of an experimental model. Available hypotheses on the association between EBV and NPC are generated from non-nasopharyngeal epithelial cell systems such as human keratinocytes or mouse epithelial cells, which may not truly represent the biological properties of nasopharyngeal epithelial (NP) cells. In this study, we report the establishment of two immortalized NP cell lines, NP69SV40T and NP39E6/E7, using SV40T and HPV16E6/E7 oncogenes. We found that NP60SV40T and NP39E6/E7 cell lines not only maintained many characteristics of normal NP cells (i.e. keratin profile and responsive to TGFbeta inhibition) but also highly responsive to one of the EBV encoded genes, LMP1. Comparative genome hybridization (CGH) analysis showed that these two cell lines contained multiple genetic alterations, some of which have been described in NPC. The immortalized NP cell lines are non-tumorigenic and exhibit anchorage-dependent growth. These cell lines may provide a possible cell model system for studying the mechanisms involved in the tumorigenesis of NPC.     

Retrovirus-mediated conditional immortalization and analysis of established cell lines of osteoclast precursor cells

Osteoclast precursor cells (OPCs) have previously been established from bone marrow cells of SV40 temperature-sensitive T antigen-expressing transgenic mice. Here, we use retrovirus-mediated gene transfer to conditionally immortalize OPCs by expressing temperature-sensitive large T antigen (tsLT) from wild type bone marrow cells. The immortalized OPCs proliferated at the permissive temperature of 33.5 degrees C, but stopped growing at the non-permissive temperature of 39 degrees C. In the presence of receptor activator of NFkappaB ligand (RANKL), the OPCs differentiated into tartrate-resistant acid phosphatase (TRAP)-positive cells and formed multinucleate osteoclasts at 33.5 degrees C. From these OPCs, we cloned two types of cell lines. Both differentiated into TRAP-positive cells, but one formed multinucleate osteoclasts while the other remained unfused in the presence of RANKL. These results indicate that the established cell lines are useful for analyzing mechanisms of differentiation, particularly multinucleate osteoclast formation. Retrovirus-mediated conditional immortalization should be a useful method to immortalize OPCs from primary bone marrow cells.     

Immortalization of rat keratinocytes by transfection with polyomavirus large T gene

Transfection of primary rat keratinocytes with the polyomavirus large T gene promotes the establishment of cell lines. The keratinocytes express the large T protein and can be continuously cultured in medium containing a low concentration of calcium. The immortalized keratinocytes retain the ability to differentiate when the calcium concentration is increased to normal levels.     

Characterization of a monoclonal antibody reactive with a glycolipid antigen expressed by tumorigenic and certain immortalized,non-tumorigenic rat esophageal epithelial cell lines

A monoclonal antibody (mAb 5G) was produced against a tumorigenic rat esophageal epithelial cell line, designated B2T. Using an enzyme-linked immunosorbent assay, immunofluorescence assay (IFA), thin-layer chromatography (TLC) and immunoperoxidase staining, it was found that mAb 5G reacted specifically with a glycolipid antigen expressed by three tumorigenic rat esophageal epithelial cell lines, and two out of the three nontumorigenic, immortalized rat esophageal epithelial cell lines tested; but did not react with primary cultures of normal rat esophageal epithelial cells or fibroblasts. mAb 5G did not bind to rat respiratory tract carcinoma cell lines, to immortalized rat tracheal epithelial cell lines, or to primary cultures of normal rat tracheal epithelial cells. In addition, mAb 5G did not react with any of the human or mouse cell lines tested. In IFA experiments, mAb 5G stained imprints prepared from in vivo propagated B2T tumor tissues, but did not react with normal rat esophageal, tracheal, lung, liver, and kidney tissues. The antigen was identified by TLC as a neutral glycolipid, consisting of two bands, withR _F = 0.45 and 0.41, which migrated in proximity to the ceramide trihexoside standard on TLC plates. Densitometric scanning of the antigen bands indicated that the tumorigenic rat esophageal cell lines possessed 50%–90% more mAb-5G-reactive antigen than the nontumorigenic esophageal cell lines. The results show that mAb 5G reacts specifically with a glycolipid antigen expressed by tumorigenic and certain non-tumorigenic, immortalized rat esophageal epithelial cell lines that might be at the late stages of transformation and early malignancy.