Describing shape dynamics in transformed cells through latent factors.

Previous studies attributed the characteristic shape changes found in cancer cells, in part, to aberrant vesicle traffic. Typically, transformed cells also rounded up. These phenomena were further investigated by measuring the shape features of cells from established lines, which represented both normal and oncogenic stages of transformation. Although conventional pattern recognition methods, applied to a combined data set from these lines, failed to reveal any new, recognizable features beyond those already known, factors did describe such features. Factors are hypothetical variables that contribute to the variance of two or more measurable variables. One factor for the cell edge, 5, was known from previous studies on correlations among the variables. Several other factors at the same level identified crucial features. Factor 4 reflected the frequency of microspikes; another factor described a knob-like structure (7). A third, factor 16, indexed the variability in projection size. Factors of the upper cell, 1 micrometer or more above the substratum, namely, 1, 2, 8, 11, 13, and 19, also described transformation-related changes. Comparing lines that modeled the development of bronchogenic carcinoma, we found a tendency for 2 (surface smoothing), 4, and 12 (rounding-up) to be changed irreversibly. Thus, factors overcame the problem of relating mathematical shape phenotypes, previously obtained based on single variables, to cell features.

Altered localization of E-cadherin and alpha-catenin in rat esophageal tumors.

An alteration in the localization of E-cadherin and its associated proteins has been observed in many epithelial neoplasms. No data exist, however, for the expression of these proteins in an animal model system for esophageal cancer or in cultured rat esophageal epithelial cell lines. The present study investigated the localization of E-cadherin and its associated protein, alpha-catenin, in rat esophageal epithelial cell lines of differing tumorigenic potential; in tumors induced after transplantation of these cell lines into syngeneic hosts; and, in esophageal tumors induced in rats by the carcinogen, N-nitrosomethylbenzylamine (NMBA). Immunofluorescent staining of the cultured cell lines revealed staining for both E-cadherin and alpha-catenin at cell-cell boundaries. Western blot analysis confirmed the membrane-bound localization of E-cadherin and alpha-catenin in the cells. However, tumors induced by these cell lines in syngeneic rats showed reduction in the expression of both E-cadherin and á-catenin in the plasma membrane of invasive epithelial cells. Immunohistochemical analysis of NMBA-induced esophageal neoplasms in rats revealed E-cadherin and alpha-catenin to be abnormally expressed in poorly differentiated tumors when compared to well differentiated tumors. These results suggest that the microenvironment may have an important role in regulating the expression of these adhesion molecules in rat esophageal epithelial cells, and that alteration in the cellular localization of E-cadherin and alpha-catenin may be indicative of tumor progression in NMBA-induced rat esophageal cancer.

Cytogenetic analyses of a murine carcinoma cell line and six metastatic derivatives with different degrees of radioresistability.

We reported that a murine carcinoma (DEN3) an its six pulmonary metastases (M2, M4C, M4D, M4E, M4F, and M6) exhibited different degrees of radioresistability (In Vitro Cell. Dev. Biol.26:222-228; 1990). While the M2, M4C, M4E, and M4F cultured cells survived up to 2.5 Gy, the cells of DEN3 and M6 tolerated up to 5.0 Gy, and the M4D cells could withstand up to 10.0 Gy of X-irradiation. In the present investigation, the cytogenetic features of these cell lines were examined: (a) to determine the degree of cytogenetic heterogeneity among these cell lines, and (b) to investigate whether any association between the cytogenetic anomaly and the degree of radioresistability could be established. Heterogeneous cytogenetic aberrations were detected in all of the above lines. Karyotype analysis of the M4D and M6 cell lines displayed both numerical and structural abnormalities. The gain and loss of chromosomal copies were observed. Structural aberrations, such as translocation and deletion appeared in both cell lines. However, correlation between the cytogenetic abnormality and the degree of radioresistability was not demonstrated except for a dramatic reduction in one or more copies of the X-chromosome that occurred in 86% and 93% of the M6 and M4D cells, respectively. The results suggest heterogeneous cytogenetic aberrations among these cell lines and a possible association between the loss of X-chromosome and radioresistability of these tumor cells.

Frameshift mutation in codon 176 of the p53 gene in rat esophageal epithelial cells transformed by benzo[a]pyrene dihydrodiol.

Mutations in the p53 tumor suppressor gene have been associated with exposure to environmental chemical carcinogens. Cultured rat esophageal epithelial cells were transformed in vitro by treatment with benzo[a]pyrene dihydrodiol (BP-DHD). A BP-DHD-transformed cell line and control cell lines were analyzed for mutations in the p53 gene and in the Ha-ras gene by single-strand conformation polymorphism analysis of polymerase chain reaction-amplified products and direct DNA sequencing. The deletion of one cytosine in codons 174-176 (TGCCCCCAC-->TGCCCCAC) of the p53 gene was found only in the BP-DHD-transformed cell line. The BP-DHD-transformed cells were highly invasive and tumorigenic when transplanted into syngeneic rats, whereas control lines either were nontumorigenic or formed epithelial cysts. BP-DHD-transformed cells and control lines were negative for mutations in the Ha-ras gene. Our results suggest that the tumorigenic potential of the BP-DHD-transformed cell line is associated with a frameshift mutation in codon 176 of the p53 gene but not with mutations in the Ha-ras gene. The G/C-rich codons 174-176 in the rat p53 gene may be specific targets for BP-DHD.

Epitope masking of rat esophageal carcinoma tumor-associated antigen by certain coexisting glycolipid and phospholipid molecules: a potential mechanism for tumor cell escape from the host immune responses.

A monoclonal antibody (mAb-5G) produced against a tumorigenic rat esophageal cell line, B2T, was shown to react specifically with a unique glycolipid antigen expressed on the cell surface of tumorigenic and certain non-tumorigenic, immortalized rat esophageal cell lines [Cancer Immunol Immunother 36: 94 (1993)]. In enzyme-linked immunosorbent assay experiments, mAb-5G reacted with crude lipid extracts prepared from B2T cells cultured in vitro, but showed very little reactivity with crude lipid extracts prepared from the same cell line passaged once in vivo, unless the antigen was separated from other lipid components by column or thin-layer chromatography (TLC). When a secondary tissue-culture cell line was established from the above B2T tumor tissues and serially subcultured in vitro, the percentage of positively stained cells was increased significantly in immunofluorescence assay. It was also demonstrated that the amount of extractable antigen was increased as the cells were subcultured in vitro up to passage 15, and stabilized thereafter. These results indicate the presence of certain lipid components in crude lipid extracts from B2T cells grown in vivo that are capable of interfering with antigen-antibody binding. On TLC plates, these interfering lipids were identified as phosphatidylcholine, phosphatidylserine, sphingomyelin and gangliosides. The interfering lipids did not bind the antibody, rather they appeared to interfere with antigen accessibility. These lipid substances may modify tumor cell surface antigen(s), thus protecting the tumor cells from host immune destruction.

Role of H-ras in the malignant progression of rat tracheal epithelial cells.

The effect of an activated H-ras oncogene on the progression of neoplasia was studied in transformed rat tracheal epithelial cells. Nude mouse tumours produced by injection of these cells exhibited a higher fraction of cells containing the mutant ras gene that did the injected cells, while a subclone that lacked the ras mutation was much less tumorigenic than parental cells. Serial passage of one cell line containing a ras mutation resulted in an increase in the fraction of ras-mutated cells, which suggests that, in this line, ras activation may confer a selective advantage in vitro as well. However, this was not seen in another ras-containing line, suggesting the importance of alternative pathways in malignant progression of rat tracheal epithelial cells.

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 non-tumorigenic, 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, with RF = 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 non-tumorigenic 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.

A monoclonal antibody produced against a rat esophageal carcinoma cell line reacts with an integrin-like molecule expressed by rat epithelial cells.

A monoclonal antibody, designated MAb-5A IIgG1), was generated against a tumorigenic rat esophageal epithelial cell line, B-2T. MAb-5A reacted with a series of non-tumorigenic and tumorigenic epithelial cell lines derived from F-344 rat esophagi or tracheas. However, the highest level of antigen expression was detected on tumorigenic rat epithelial cell lines. A trace amount of antigen was detected in primary cultures of normal rat epithelial cells derived from either esophagi or tracheas. MAb-5A did not react with rat fibroblasts. MAb-5A reacted with B-2T derived tumor tissues propagated in vivo but showed only slight reactivity with normal rat esophageal epithelial tissues. Cell surface radioiodination, extraction and immunoprecipitation experiments were conducted to characterize the antigen molecule. Analysis of the immunoprecipitates by SDS-PAGE and autoradiography revealed two protein bands with mobilities corresponding to approximately 140 and 120 kDa, respectively. Under reducing conditions the 140 kDa band shifted to approximately 120 kDa whereas the 120 kDa band shifted upward to approximately 130 kDa. The non-reduced/reduced mobilities of these bands suggest that they may be members of the integrin family of matrix receptors. A polyclonal antibody to the beta 1 subunit immunoprecipitated two similar bands detected by MAb-5A, further suggesting that the antigen complex may be related to members of the integrin superfamily.

Biological heterogeneity and radiation sensitivity of in vitro propagated lung metastatic lines originated from a transplantable squamous cell carcinoma of BALB/c mouse.

Seven cell lines established from a diethylnitrosamine (DEN)-induced forestomach carcinoma (DEN3) of a BALB/c mouse and its six pulmonary metastatic foci were used to study the biological and functional diversity of tumor cells. DEN3 is a highly tumorigenic line capable of forming lung metastases readily. Six metastatic nodules were isolated from the lungs of syngeneic mice and six cell lines were established. The cell lines differed in characteristics such as tumorigenicity, metastatic capability, and in vivo and in vitro growth properties. Radiation sensitivity of these cell lines was examined by exposure, at near confluency stage of in vitro growth, to doses of 2.5 to 50 Gray (Gy) X-rays (1 Gy = 100 rads). Shortly after exposure (approximately 5 min), the cells were harvested and 10(5) cells were cultured or inoculated into syngeneic mice, or both. Growth of three of the six cell lines tested was prohibited by 5 Gy. However, some populations from the other cell lines were able to survive 5 or 10 Gy. Progenies of the cells that survived primary radiation exposure after several in vitro passages were able to withstand another exposure of the same magnitude but not a higher dose. The X-rayed survivor cells also maintained their tumorigenic potential.

Comparative properties of untreated and N-nitrosobenzylmethyl-amine-transformed rat esophageal epithelial cell lines.

A culture system utilizing rat esophageal epithelial cells has been developed. Four normal and eight N-nitrosobenzylmethylamine-treated lines were compared with respect to chromosome number, anchorage-independent growth in agarose, and tumorigenic potential in syngeneic rats. All cell lines were aneuploid with nine in the near-tetraploid range and three in the near-diploid range. No relation between tumorigenic potential and chromosome number or structure was apparent. Similarly, anchorage-independent growth in agarose did not correlate with tumorigenic potential. Three of the 12 immortalized lines (two carcinogen-treated and 1 untreated) induced well-differentiated squamous cell carcinomas in syngeneic rats. These tumors had weak metastatic potentials suggesting that tumorigenic potential and metastatic ability are separately controlled. These cell lines will be useful for the investigation of factors involved in the conversion of immortalized rat esophageal epithelial cell lines to lines of high metastatic potential.

Growth inhibition and DNA damage induced by benzo[a]pyrene and formaldehyde in primary cultures of rat tracheal epithelial cells.

The effects of benzo[a]pyrene (BAP) and formaldehyde (HCHO), alone and combined, on cell growth and DNA damage were determined in primary cultures of rat tracheal epithelial cells dissociated from rat tracheas. Cell cultures treated with 25 microM BAP for 24 h or 200 microM HCHO for 90 min did not have a marked reduction in cell growth. However, their combined treatment reduced cell growth by 60% of control when cultures were exposed to BAP followed by HCHO as well as the reverse order. None of these treatments significantly decreased cell viability as judged by dye exclusion, nor did they enhance cell terminal differentiation as measured by cornified envelope formation. Alkaline elution analysis of DNA damage detected both DNA-protein crosslinks (DPC) and DNA single-strand breaks (SSB) as a result of HCHO treatment, whereas BAP treatment caused only SSB. While HCHO-induced SSB were repaired within 2 h, BAP-induced SSB were detected 3 days after treatment. Combined treatment of cell cultures with BAP followed by HCHO resulted in more SSB than was obtained from either agent alone, but less DPC than was detected from HCHO alone. The increased number of SSB obtained from this combined treatment may be related to the marked enhancement of carcinogenesis observed in earlier in vivo-in vitro studies.

Enhancement of lung tumor colony formation by treatment of mice with monoclonal antibodies to pulmonary capillary endothelial cells.

Two rat monoclonal antibodies, 34A and 201B, have been isolated and shown to bind preferentially to capillary endothelial cells in the lung. Administration of these antibodies to mice increases the number of lung colonies derived from i.v. injection of tumor cells. The antibodies increase lung colonization in C57BL/6 mice following i.v. injection of B16-F10 melanoma cells and in BALB/c mice following injection of line 1 lung carcinoma cells. Neither 34A nor 201B monoclonal antibody binds to B16 melanoma or line 1 carcinoma and so must exert its effect by interaction with endothelial cells. Antibodies injected i.v., s.c., or i.p. are active from 1 h to 1 wk if injected before cell injection. The effect is optimal when 0.1 ml of ascites fluid containing 120 micrograms of antigen binding capacity of both MoAbs 34A and 201B is injected. Significant damage to endothelial cells could not be documented by histopathological examination at the light microscope level or by protein leakage into the air space as measured by lung lavage. However, electron micrographs taken 3 h after monoclonal antibody injection show minor damage to endothelial cell membranes throughout the lung with some areas of mild edema. The increased colonization may be mediated by this subtle damage to endothelial cells, or antibody interactions with endothelial cells may trigger secondary reactions such as altered expression of growth factors.

Increase in immunogenicity with concomitant loss of tumorigenicity of respiratory tract carcinomas during in vitro culture.

Cell lines were established in vitro from respiratory tract carcinomas induced in rats by carcinogenic, polycyclic hydrocarbons. Propagation of the carcinoma lines in vitro lead to a progressive decrease in tumorigenicity. Tumor transplantation studies in X-irradiated, immunosuppressed recipients and in immunologically reconstituted recipients suggested that the cells are rejected because of their immunogenicity, since a high incidence of tumors was observed in X-irradiated recipients but not in normal or X-irradiated, reconstituted recipients. When immunologically competent rats were immunized with cells from an in vitro tumor line, strong tumor transplantation resistance resulted. Similar immunization with the corresponding in vivo tumor line caused very little if any protection, and immunization with a non-cross-reacting sarcoma line grown in vitro did not produce immunological protection against carcinoma cell lines. A single in vivo passage of the in vitro-adapted tumor line in immunosuppressed recipients fully restored tumorigenicity. The increase in immunogenicity of carcinomas cultured in vitro appears to involve preexisting angigens indigenous to the carcinomas rather than new antigens acquired during tissue culture, such as antigens related to retroviruses, mycoplasmas, or heterologous serum.

Detection of circulating tumor antigens in mice carrying a highly metastatic pulmonary squamous--cell carcinoma.

Recently we reported that the highly metastatic MSC-10 (mouse squamous carcinoma) is incapable of inducing transplantation immunity. Studies reported here were undertaken to determine whether or not the tumor is devoid of tumor-associated antigen. We found that sera from MSC-10 tumor-bearing mice contain soluble protein antigens which react with rabbit antisera made against the MSC-10 tumor, as demonstrated by immuno-diffusion. Such proteins were not detected in the sera of normal mice or mice bearing fibrosarcomas. A close temporal relationship was demonstrated between the appearance of circulating antigens and the occurrence of lung metastases. Protein components serologically indistinguishable from the circulating antigens were isolated from tumor cells. The molecular weight of these proteins is between 30,000 and 100,000 daltons. Studies with antisera to mouse leukemia virus showed that hte MSC-10 tumor antigens are not viral proteins. The lack of immunogenicity of this tumor for syngeneic hosts as well as its high metastatic activity may be due to the early appearance of soluble antigens in the circulation.

Demonstration of cellular and humoral immunity to transplantable carcinomas derived from the respiratory tract of rats.

Previous studies with respiratory tract tumors in mice have suggested that such tumors are not immunogenic or are only weakly so. To determine whether this is a general characteristic of neoplasias found in the airways of rodents, we investigated seven transplantable carcinomas in rats, six of which originated from tracheal epithelium and one of which orginated from the distal lung. These carcinomas were all of the squamous type and were induced by three different carcinogenic polycyclic hydrocarbons. All of the tumors were shown to be immunogenic, capable of mobilizing cellular and humoral immune responses in isogenic hosts upon immunization. This was demonstrated by induction of transplantation resistance, by Winn's neutralization test, and by the detection of antibodies in the sera of tumor-immune hosts by two independent methods (antibody-dependent cytotoxicity and antibody-binding test). The degree of immunogenicity varied among the tumor lines. The most metastatic tumor was clearly the least immunogenic. The relationship between carcinogenic insult and immunogenicity, as well as the possible nature of the tumor-associated antigens involved, is discussed.

Influence of bacteriological media constituents on the reproduction of Salmonella enteritidis bacteriophages.

Three different Salmonella enteritidis phages were isolated and purified from raw sewage by agar-layer technique. The sensitivity of the host organisms toward phages was changed when they were grown on different bacteriological media. The effect of single components of the medium on phage reproduction was determined by the omission of that substance from the medium. CaCl2,MgSO4, and glycerol each had a pronounced stimulatory effect on the phage reproduction, while bile salts had a profound inhibitory effect. The inhibitory effect of bile salts on phage growth was much greater on one strain of Salmonella enteritidis than on the other.

Non-immunological enhancement of tumour transplantability in x-irradiated host animals.

MSC-10 tumour cells (derived from a chemically induced pulmonary squamous-cell carcinoma in DBA/2 mice) were inoculated intramuscularly into thymectomized, X-irradiated isogeneic mice, either 48 h or 6 weeks after thymectomy and X-irradiation. Normal mice and immunologically reconstituted mice served as controls. A marked enhancement in frequency of tumour takes was observed in all groups of animals inoculated with tumour cells 48 h after whole:-body X-irradiation, whether thymectomized, immunologically reconstituted or not. The TD50 decreased to less than 1/10 of that observed in unirradiated controls. When mice were inoculated with tumour cells 6 weeks after X-irradiation, the incidence of tumour takes was similar to that of unirradiated controls, including the thymectomized-irradiated group, which was still severely immunodeficient as measured by antibody formation and skin graft rejection. The experiments indicate that whole-body X-irradiation creates a condition that favours tumour cell survival or growth. This "permissive state" exists only shortly after X-irradiation and is not correlated with the host's level of immunocompetence.

Increase in immunogenicity of a pulmonary squamous-cell carcinoma, propagated in vitro.

The chemically induced, non-immunogenic lung squamous-cell carcinoma (MSC-10) propagated in vitro gradually loses tumorigenicity in immunocompetent hosts with increasing in vitro passage. This was found to be related to an increase in antigenicity, since immunosuppressed hosts (thymectomy plus 600 rads whole body X-irradiation) supported the growth of tumor cells, whereas immunocompetent recipients did not. The antigens involved in rejection are not heterologous serum proteins present in culture media since the cell line grown in isologous serum is also rejected. Immunization with the in vitro tumor line partially protected against the parental in vivo line, therefore the antigens involved must be present on both tumor lines. Inoculation of the cultured cell line into normal or immunosuppressed hosts produced tumors with the same histological characteristics as those of the in vivo tumor line. We concluded that by in vitro culture the weakly antigenic carcinoma becomes more immunogenic and thereby capable of inducing transplantation resistance. The cultured tumor cells retain their antigenic specificity and histologic characteristics while increasing their antigenic potency.