Cytogenetic studies of esophageal carcinoma cell lines.

Although the incidence of cancer of the esophagus is low in the United States, the prognosis of patients with this malignancy is poor, especially when metastases exist. More research concerning the biological characteristics of this tumor is necessary to permit more effective treatment and to determine the etiology. We successfully studied cytogenetically 14 short- and long-term cell lines derived from esophageal carcinoma to determine whether these tumors have nonrandom, unique chromosomal abnormalities. Our results showed that the tumor cells had chromosome numbers clustering around a modal number that varied according to the cell line. The presence in the primary explant of extensive numerical and structural abnormalities involving every chromosome including the sex chromosomes indicate that these abnormalities occur early in the malignant cells. The chromosomes most frequently involved in the structural abnormalities were 1, 9, and 11, each occurring in 13 of the 14 lines, and of three found in 12 of the 14 lines. The major aberrations resulted in deletions of portions of these chromosomes. The most frequent breakpoints for these abnormalities occurred at 3p14, 11q11q12; and 9q11q12 as well as in the centromeric regions of all the acrocentric chromosomes. Another unusual chromosomal marker found in three lines (HCE-1, HCE-3, and HCE-5) was a homogeneously staining region (HSR) that occurred as an extension on 11q12.

Effects of tumor promoters and cocarcinogens on growth and differentiation of cultured human esophageal epithelial cells.

The acute effects of 12-O-tetradecanoylphorbol-13-acetate [(TPA) CAS: 56937-68-9], T-2 toxin (CAS: 21259-20-1), capsaicin (CAS: 404-86-4), cigarette smoke condensate (CSC), and ethanol (CAS: 3807-77-0) were examined in secondary cultured human esophageal epithelial cells in serum-free LHC-8 medium. Effects were evaluated by morphology and measurement of clonal growth rate (population doublings per day), cross-linked envelope (CLE) formation, and the enzymatic activities of ornithine decarboxylase (ODC) and plasminogen activator (PA). All compounds tested were inhibitory to clonal growth; concentrations causing 50% growth inhibition were estimated as 10 nM TPA, 6 nM T-2 toxin, 40 microM capsaicin, 8 micrograms CSC/ml, 540 mM ethanol, and 0.8 microgram CSC/ml with 220 mM ethanol. None of the compounds tested induced CLE formation, although calcium ionophore (A23187) could induce CLE in at least 60% of the cells. TPA (10 and 100 nM) decreased the ODC activity of cells, and capsaicin (100 microM) induced ODC by 220%. TPA (1-100 nM) and capsaicin (100 microM) also induced PA activity. Slight increases in ODC activity by CSC (10 micrograms/ml), CSC (1 microgram/ml) with ethanol, and T-2 toxin (1 nM) were observed, but PA activity was not affected by these compounds. The results indicated that the response of human esophageal epithelial cells to TPA is both similar to and different from that reported for human epidermal and bronchial cells in vitro. Enhancement of PA activity and decrease in ODC by TPA are found in all three human epithelial cell types. However, these changes are not associated in esophageal cells with increased CLE formation as reported in studies with the use of bronchial and epidermal epithelial cells. The results from these acute studies provide the basis for designing in vitro carcinogenesis investigations using these agents.

Human esophageal carcinoma cells have fewer, but higher affinity epidermal growth factor receptors.

Squamous cell carcinomas have recently been shown to contain increased numbers of epidermal growth factor (EGF) receptors. Since EGF has an important role in epithelial growth and differentiation, it is possible that modulation of its receptor may have an important role in neoplasia. In an attempt to further explore the relationship of EGF receptor expression to malignant transformation, we examined 14 squamous cell carcinoma cell lines of the esophagus for the number and affinity of EGF receptors. Seven cell lines were newly isolated by this laboratory and recently characterized. The seven additional cell lines were obtained from Japan (4 cell lines) and South Africa (3 cell lines). Surprisingly, we found that esophageal carcinomas contained lowered quantities of surface EGF receptors (2- to 100-fold) and that the affinity of the EGF receptor was increased (6- to 100-fold) when compared to normal esophageal epithelial cells. Moreover, the biologic response of esophageal carcinoma cells to EGF differed markedly from that of other squamous cell tumor cells exhibiting elevated numbers of receptors, such as A431 and SCC-15. Human esophageal carcinoma cells were maximally stimulated by the addition of 5 ng/ml of EGF, similar to normal esophageal keratinocytes, but in contrast to normal cells were not inhibited by the higher concentrations tested (up to 40 ng/ml). On the other hand, addition of any EGF to the medium (beyond that normally present in serum) was found to dramatically inhibit the growth of A431 and SCC-15 cells. Our findings indicate that squamous cell neoplasia is not dependent upon increased numbers of cell surface EGF receptors, that EGF receptor number may have a determinant role in EGF cell toxicity, and that the stimulatory response of cells to EGF may reflect a complex function of EGF receptor number, affinity, and occupancy.

Growth and differentiation of human esophageal carcinoma cell lines.

Human esophageal carcinoma cell lines (8 cell lines) differed from their normal counterpart in terms of their morphological appearance, growth properties, and the expression of certain differentiated functions, namely keratin proteins and cross-linked envelopes. In contrast to normal human esophageal keratinocytes, the carcinoma cells were pleomorphic and tended to pile up in an unorganized fashion. When grown under optimal growth conditions the carcinoma cells generally grew to a higher saturation density than their nontransformed counterpart; their generation times were variable. Transformed cells grew better under stringent growth conditions (e.g., decreased serum and no additional growth factors except hydrocortisone) than did nontransformed human esophageal keratinocytes but their growth was still restricted under these conditions. The carcinoma cells retained a requirement for a 3T3 feeder layer when grown at clonal densities (5 X 10(3) cells/60-mm dish) but could be passaged and maintained without a feeder layer if plated at higher than clonal densities (10(5) cells/60-mm dish). All cell lines grew in an anchorage-independent fashion in soft agarose although the colony forming efficiency and size of the colonies varied among the different cell lines. Not all anchorage-independent cell lines were tumorigenic. Tumorigenic potential was greatly augmented by the use of cell lines derived from soft agarose selected clones. Altered expression of keratin proteins and cross-linked envelopes was observed in the carcinoma cell lines and generally reflected those changes seen in primary esophageal carcinomas. In two cell lines (HCE-4 and HCE-6), the synthesis of the Mr 44,000 (analogous to Rheinwald's Mr 40,000 keratin) and 52,000 keratins was suppressed coincident with the appearance of the 67 Kd keratin in tumors derived from these cell lines. These keratin patterns were once again reversed in cell lines recultured from these tumors, suggesting that the expression of these specific keratins is subject to extrinsic growth regulation. Another feature of terminal differentiation in keratinocytes, cross-linked envelope formation, was found to be significantly altered (reduced) in most but not all human esophageal carcinoma cell lines.

Keratin expression of both chemically and virally transformed human epidermal keratinocytes during the process of neoplastic conversion.

Human epidermal cells, despite being 'immortalized' or 'transformed' by combinations of either oncogenic virus (SV40, adenovirus 12 or Kirsten murine sarcoma virus) or chemical carcinogen (N-methyl-N'-nitro-N-nitrosoguanidine or 4-nitroquinoline-1-oxide) exhibited similar keratins (although quantitatively reduced) to that of control cells when grown in vitro. However, athymic nude mouse tumors derived from such cells exhibited suppression of the 52, 56 and 58 kd (basic type II) keratins and a predominance of small-sized (40-48 or 50 kd) (acidic type I) keratins. The synthesis of these specific keratins was resumed following re-establishment of cell lines in culture. These results suggest that the changes in keratin protein profiles frequently exhibited by human carcinomas represent a component of the pleomorphic transformed phenotype which can be uncoupled from neoplastic growth.

Intermediate filament and cross-linked envelope expression in human lung tumor cell lines.

Human lung tumor cell lines established from the major histological types of lung cancer were examined by immunofluorescent staining techniques for their patterns of intermediate filament (keratin, vimentin, and neurofilament triplet protein) expression. All cell lines examined, both small cell lung carcinoma (SCLC) and non-SCLC (squamous cell carcinoma, adenocarcinoma, large cell carcinoma, and mesothelioma) contained keratin, consistent with their epithelial derivation. These lung carcinoma cell lines also expressed vimentin, the characteristic intermediate filament of mesenchymal cells in vivo. In light of the proposed neuroectodermal origin of SCLC, cell lines were also studied for neurofilament expression. Two of four SCLC tumor cell lines, as well as non-SCLC cell lines, showed no reactivity with antibodies to neurofilament triplet protein. Two of the SCLC cell lines stained weakly with anti-neurofilament antibody. Examination of specific keratin patterns in human lung tumor cell lines by selective immunoprecipitation with keratin antiserum and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that small-sized keratin proteins (Mr 44,000 to 52,000) were present in cell lines derived from SCLC and non-SCLC types of lung cancer. Tumor cell lines exhibiting squamous differentiation by light microscopic criteria (i.e., intracellular keratin, intercellular bridging, "pearl" formation, and/or individual cell keratinization) also displayed a preponderance of intermediate-sized keratins (Mr 57,000 and 59,000) and exhibited another feature of terminal keratinocyte differentiation (cross-linked envelope formation). Mesothelioma cell lines had varying keratin profiles. The presence of keratin proteins in all SCLC cell lines examined argues against a neuroectodermal origin for these tumors and is consistent with the notion that these tumors arise from a common bronchial "stem cell," similar to that from which other types of bronchogenic carcinomas arise.

Keratin proteins and carcinoembryonic antigen in synovial sarcomas: an immunohistochemical study of 24 cases.

Twenty-four synovial sarcomas were examined for the presence of keratin proteins by an indirect immunoperoxidase method with paraffin-embedded tissues. Keratin proteins were identified in 16 of 24 cases (67 per cent). Both the pseudoglandular and spindle cell areas of all eight of the biphasic synovial sarcomas and the spindle cells of eight of the 16 monophasic synovial sarcomas contained keratin proteins. In spindle cell areas, staining was observed in single cells and small cords and clusters of cells in the absence of cleft formation or other evidence of a pseudoglandular component. The predominant cytologic staining pattern in all cases was peripheral, with localization of staining to the cell membrane or adjacent areas, but diffuse and focal cytoplasmic staining patterns were also observed. No staining for keratin proteins was seen in 101 control cases, including 52 sarcomas of various types. Carcinoembryonic antigen was also identified in four of the 24 synovial sarcomas by an indirect immunoperoxidase technique. The identification of keratin proteins may be helpful in the pathologic diagnosis of synovial sarcoma, particularly the spindle cell monophasic variant.

Aberrant expression of keratin proteins and cross-linked envelopes in human esophageal carcinomas.

When compared to normal esophageal epithelium, marked alterations in keratin protein and cross-linked envelope expression were found in human esophageal carcinomas. Examination of the pattern of keratin proteins extracted from either several primary esophageal tumors or carcinomas xenotransplanted in nude mice revealed a dramatic reduction in the amount of keratin protein, especially in the Mr 52,000 to 61,000 range. In seven of eight of the primary tumors, the major Mr 52,000 and Mr 61,000 esophageal keratins were not detected, and the remaining tumor exhibited a marked reduction in these two keratins. The major Mr 57,000 and minor Mr 59,000 esophageal keratins were found in varying but reduced amounts in the different tumors. The major Mr 57,000 keratin seemed to be the most conserved keratin of this intermediate-molecular-weight keratin class (Mr 52,000 to 61,000). In contrast, the lower-molecular-weight keratins (Mr 46,000 to 50,500) were usually conserved in the carcinoma cells and were present at levels approximating that of the nontransformed counterpart. The minor Mr 37,000 and 44,000 keratins from normal esophageal epithelium were retained in the tumor cells but often in reduced amounts. The expression of another differentiated function, cross-linked envelopes, in the carcinoma cells varied from unimpaired to severely restricted capacity to form envelopes. In conclusion, specific alterations in keratin protein and cross-linked envelope expression were found in human esophageal carcinomas.

Keratin proteins in human lung carcinomas. Combined use of morphology, keratin immunocytochemistry, and keratin immunoprecipitation.

Light-microscopic immunocytochemistry and electron microscopy demonstrated that adenocarcinomas (AC) and squamous cell (epidermoid) carcinomas (SCCs) of human lung contained keratin proteins in the form of tonofilament bundles. However, moderately differentiated (md) SCCs contained abundant keratin, whereas poorly differentiated (pd) SCCs and all ACs contained lesser amounts. Lung tumors with the diagnosis of AC or SCC, as defined by WHO criteria, were also analyzed by immunoprecipitation techniques for the presence of keratin proteins. Regardless of the degree of tumor differentiation, SCCs contained a 44 kd keratin which was lacking in ACs. Interestingly, normal bronchial epithelium also contained the same 44 kd keratin. In addition, as SCCs became more differentiated, they exhibited even greater differences in the profile of synthesized keratins. Specifically, the relative abundance of the intermediate-sized keratins (57 and 59 kd) was increased in the md SCCs. Although keratin protein patterns appear to be a valuable adjunct in distinguishing AC from SCC, their usefulness as a diagnostic tool will require survey of a larger number of poorly differentiated tumors.

Immunocytochemical localization in normal and transformed human cells in tissue culture using a monoclonal antibody to the src protein of the Harvey strain of murine sarcoma virus.

Using a rat monoclonal antibody directed against the p21 src protein of the Harvey strain of Murine Sarcoma Virus (MSV), we have examined the reactivity of human cells in tissue culture using immunofluorescence and electron microscopy. Qualitative results indicated that untransformed mouse and human fibroblastic cells have undetectable amounts of p21; these levels were greatly increased after transformation with Harvey MSV. A group of human tumor cell lines adapted to tissue culture were examined and almost all of the epithelial tumor lines showed significant localization with this antibody. Notable exceptions were two melanoma cell lines which were negative for p21 by immunofluorescence. When normal human epithelial cells derived from esophageal or foreskin epithelium were examined, the antibody showed significant reactivity with subconfluent growing cells. After the normal cells were allowed to become quiescent, the reactivity with this antibody decreased. All of the localization seen by fluorescence was in a distribution consistent with the previously demonstrated location of p21 scr on the inner aspect of the plasma membrane. Electron microscope localization showed labeling for this antigen on the inner surface of the plasma membrane in both transformed mouse cells and in the human tumor cell lines MCF-7 and HTB-2 (RT4). These results suggest that the amounts of p21-like proteins detectable in human epithelial tumor cells do not necessarily reflect their malignant potential, but may be related to their epithelial nature. The loss of detectable localization at quiescence suggests that p21 levels decrease when these epithelial cells stop growing, and raises the possibility that an analog of p21 may be used by these human epithelial cells to regulate cell growth.

Tissue-specific expression of keratin proteins in human esophageal and epidermal epithelium and their cultured keratinocytes.

In contrast to the simplified keratin content of bovine, rabbit, and rat esophageal epithelium (composed mainly of a 57 and 46 or 51 kD keratin, depending on the animal species), human esophageal epithelium contained a quantitatively different array of keratin proteins, ranging in molecular weight from 37 to 61 kD. The pattern of keratin proteins from human esophageal epithelium differed qualitatively and quantitatively from that of human epidermis. Human esophageal epithelium lacked the 63, 65, and 67 kD keratins characteristic of human epidermis, consistent with the absence of a granular layer and an anucleate stratum corneum. Moreover, human esophageal epithelium contained a distinctive 61 kD keratin protein which was either not present or present in only small amounts in human epidermis and variable amounts of a 37 kD keratin. Whereas the 56, 59, and 67 kD keratins were the most abundant keratins in human epidermis, the 52, 57, and 61 kD keratins predominated in human esophageal epithelium. During in vitro cultivation, both human epidermal and esophageal keratinocytes produce colonies which are stratified, but the morphologic appearance of these cultured epithelia differs. Only cultured human epidermal keratinocytes contain keratohyalin granules in the outermost layers and a prominent 67 kD keratin on immunoprecipitation. Otherwise the keratin contents appear similar. In conclusion, human esophageal epithelium exhibited intertissue and interspecies differences in the pattern of keratin proteins. During in vitro cultivation, human esophageal keratinocytes retained some aspects of their distinctive program of differentiation.

Localization of keratin proteins in the human epidermis by a postembedding immunoperoxidase technique.

Individual keratin species were localized ultrastructurally to the tonofilaments and tonofibrils of the human epider-misusing a postembedding immunoperoxidase method. Low molecular weight keratin (45 kD) was localized primarily to the stratum basale and displayed a fine cytoplasmic staining of individual tonofilaments and some tonofibrillar staining. Higher molecular weight keratins (55 and 63 kD) were found predominantly in the suprabasal, differentiated squamous epithelial cells, staining tonofibrils with a web-like pattern. The technique utilized is suitable for studying the distribution of keratin proteins in normal and disease states.

Differentiation of human epidermal cells transformed by SV40.

Human epidermal cells were transformed with DNA from wild-type SV40 virus or with DNA from a temperature-sensitive A mutant (tsA209). The SV40-transformed cells differed from nontransformed cells in their morphologic appearance, growth properties, and expression of certain characteristics associated with differentiation. The transformed cells were more variable in size and shape than their nontransformed counterparts and were less stratified and less keratinized. While the growth properties of the cells were similar under optimal growth conditions, the transformed cells could be propagated under stringent growth conditions that did not support the growth of nontransformed human epidermal cells. The transformants still required a 3T3 feeder layer for growth, remained anchorage dependent as assayed in soft agar, and were not tumorigenic in athymic nude mice. The expression of certain differentiated functions of the human epidermal cell, the presence of keratins and cross-linked envelopes, was decreased in the transformed cells, and these functions could be restored at the nonpermissive temperature in the tsA209 transformed cells.

Keratin alterations during embryonic epidermal differentiation: a presage of adult epidermal maturation.

Differentiation of the epidermis during embryonic rabbit development was found to be accompanied by dramatic changes in keratin proteins. Immunofluorescent labeling with keratin antiserum revealed that the undifferentiated epithelium of 12-d embryos was already committed to making keratin proteins. At 18 d of embryogenesis, the epithelium contained keratin proteins in the molecular weight range of 40,000-59,000. The stratification of the epithelium into two cell layers at 20 d of development coincided with the appearance of a 65-kdalton keratin. When a thick stratum corneum developed at 29 d, several additional keratins became prominent, most notably the large keratins (61- and 64-kdalton) and a 54-kdalton keratin. In addition, the 40-kdalton keratin, which had been present in earlier embryonic epidermis, disappeared. Newborn epidermis resembled that of a 29-d embryonic epidermis, with the exception of the appearance or increase in concentration of two more keratin species (46- and 50-kdalton). In vitro culturing of keratinocytes from 12- and 14-d embryonic skin demonstrated that these cells contained essentially the same keratin profiles as the undifferentiated epithelium of 18-d embryos (40-59 kdalton). Keratinocytes grown from older embryos contained increased amounts of keratin, similar to the in vivo situation, but did not synthesize the high molecular weight keratins. The changes observed during embryonic epidermal differentiation appear to be recapitulated during the sequential maturation steps of adult epidermis.