Disorders of the actin cytoskeleton in transformed epithelial cells

The actin cytoskeleton of 8 transformed epithelial cell lines was studied using electron microscopy of platinum replicas. Seven of these lines belonged to the IAR series of rat liver epithelial cells, being at different stages of neoplastic progression. One cell line (FBT) was derived from the epithelium of bovine fetal trachea. The extent of actin cytoskeleton alteration in cell lines studied has been shown to correlate with other signs of neoplastic transformation. Among various actin-containing cell structures (microfilament bundles, actin meshwork at active edges, cell-cell adherence junctions, and endoplasmic microfilament sheath) the latter was the most sensitive to transformation. The loosening of the sheath and the alteration of its fine structure were observed in all the cell lines. The degree of these changes increased in the following order: FBT; non-tumorigenic IAR lines; IAR lines transformed in vitro; IAR lines obtained from the latter by single or double selection in vivo. The alteration of sheath was the only disturbance of actin cytoskeleton in FBT cells, whereas in other groups of epithelial cell lines some other changes occurred. These involved disruption of actin-containing intercellular junctions, the cell polarization accompanied by progressive shortening of length of the cell active edge containing actin meshwork, and disappearance or reorganization of microfilament bundles.     

Neuronal differentiation is accompanied by NSP-C expression

Neuroendocrine-specific protein (NSP) reticulons are expressed in neural and neuroendocrine tissues and cell cultures derived therefrom, while most other cell types lack NSP-reticulons. Three major subtypes have been identified so far, designated NSP-A, NSP-B, and NSP-C. We have investigated the correlation between the degree of neuronal differentiation, determined by morphological and biochemical criteria, and NSP-reticulon subtype expression. For this purpose, several human neuroblastoma cell lines, exhibiting different degrees of neuronal differentiation, were examined immuno(cyto) chemically. It became obvious that the expression of NSP-C, as detected by immunofluorescence microscopy and Western blotting, is most prominent in cell lines with a high degree of neuronal differentiation, such as LA-N-5. Such highly differentiated cells also express other neural and neuroendocrine markers, such as neural cell adhesion molecule (NCAM), neurofilament proteins, synaptophysin, and chromogranin. NSP-A was observed in all cell lines to a different extent. However, no clear correlation was observed with the degree of neuronal differentiation as defined by other neuronal and neuroendocrine markers or morphology. NSP-B could not be detected. The induction of neuronal differentiation with nerve growth factor, dbcAMP, and retinoic acid in the rat pheochromocytoma cell line PC12 and the human teratocarcinoma cell line hNT2, respectively, induced the expression of NSP-A and NSP-C in these cell lines parallel to the induction of neurofilament protein expression. It is concluded that NSP-C expression, in particular, is strongly correlated with neuronal differentiation.     

The T cell repertoire to a multideterminant antigen. Clonal heterogeneity of the T cell response, variation between syngeneic individuals, and in vitro selection of T cell specificities.

The specificity of lysozyme-induced T cell proliferative responses by individual B10.A mice was compared by using a panel of three peptides. A surprising degree of variation in the focus of the responses was observed among individual animals, both in the newly isolated lymph node cell populations and in long term T cell lines. The responses to each determinant after hen egg-white lysozyme immunization were not equal and in examining the mice as a group some determinants tended to be dominant. However, despite each animal favoring a restricted number of determinants, the responding T cell populations were still highly heterogeneous. The data suggest that many determinants are involved in the response to the whole Ag. The role of one or more dominant determinants can be overestimated because the degree of heterogeneity in long term T cell lines appears to be less than in freshly obtained lymph node cells, indicating that a process of in vitro selection occurs. We observed that the T cells responsive to one peptide, 46-61, appeared to have a selective advantage in vitro culture.     

Differential sensitization of human tumor cells by ara-A to X irradiation and its relationship to inherent radioresponse.

We have previously shown that pretreatment of plateau-phase cultures of human tumor cells with ara-A can markedly sensitize them to the cytotoxic effects of X irradiation; the degree of sensitization varied in two different cell lines. The present study was undertaken to determine whether variability in radiosensitization by ara-A occurs at random in human tumor cell lines or if it is related to their intrinsic radiosensitivity (human tumor radioresponse). The interaction between ara-A and X irradiation was examined in plateau-phase cultures of early-passage tumor cell lines of varying radioresponse (D0 range 0.85-3.15 Gy) subcultured immediately after irradiation to measure survival. In six of the eight cell lines studied, pretreatment with ara-A greatly enhanced the lethal effects of X irradiation in a concentration-dependent fashion. Little or no effect was observed in the two radiosensitive cell lines. When ara-A sensitization was plotted as a function of D10 or D, a linear relationship was observed. These data suggest that pretreatment with ara-A is effective in sensitizing radiation-resistant human tumor cells to the lethal effects of X rays, and that this phenomenon may be dependent upon inherent tumor cell radiosensitivity.     

Membrane associated antigens of human malignant melanoma V: Serological typing of cell lines using antisera from nonhuman primates

Summary Antisera against various melanoma cell lines were raised in nonhuman primates (Cercopithecus aethiop.). After exhaustive absorption with AB Rh + red blood cells and pooled platelets from about 200 donors the sera were still reactive to various degrees in the microimmune adherence test with other melanoma lines, with embryonic fibroblasts, and with non-melanoma lines. As proven by absorption experiments, the main-specificity of the antisera was not directed against components of the fetal calf serum used for cell culture or against mycoplasma grown from commercial fetal calf serum. In addition, no cross-reactivity was observed with Bacillus Calmette-Gérin, and in blocking experiments no reactivity against extracts of common bacterial antigens or mixed molds was detected. Absorption with embryonic fibroblasts or embryonic tissue showed that the reactivity of most antisera was directed against melanoma-associated antigens expressed also on fetal tissue. It was not possible to determine whether the remaining reactivity on some cell lines was melanoma-specific or directed against fetal antigens not contained in the fetal material used for absorption. Cross-absorption of antisera with other melanoma cells revealed that various cell lines express different patterns of tumor-associated antigens with no, or only partial, overlap. The cross-absorption experiments made it possible to type the cell lines according to their surface antigens and arrange the cell lines in order according to the degree of mutual antigenic relationship.     

Biological properties of human melanoma cells in culture.

Three human melanoma cell lines derived from one primary and two metastatic tumors from three different patients were characterized for growth properties usually associated with malignant transformation; these include cell morphology, growth rate, saturation density, growth in semisolid media, colony-forming ability on contact-inhibited monolayers of normal fibroblasts and epithelial cells, and tumorigenicity in immunosuppressed mice. Variations in expression of aberrant properties were evident among the lines. One of the metastatic lines satisfied all the parameters of malignancy tested and the other showed a number of these properties, whereas the primary essentially fulfilled only one. These results suggest that cultured melanoma cells reflect the clinical variability often observed among melanoma patients and that metastatic melanoma seems to display a higher degree of malignant transformation than the primary.     

Effects of transforming growth factor β on growth of human mammary epithelial cells in culture

Summary Normal human mammary epithelial cells (HMEC) from different individual reduction mammoplasty specimens were all growth inhibited, and showed a flattened, elongated morphology in response to human recombinant transforming growth factor β1 (TGFβ). The degree of growth inhibition varied among specimens, but none of the normal HMEC maintained growth in the continued presence of TGFβ. The degree of growth inhibition also varied with cell age in vitro, cells closer to senescence being more sensitive. TGFβ sensitivity was additionally assayed in two established cell lines derived from one of the reduction mammoplasty specimens after exposure to benzo(a)pyrene. Although varying degrees of growth inhibition and morphologic changes were observed in the cell lines, both lines contained populations that maintained active growth in the presence of TGFβ. Subclones of these lines demonstrated a great plasticity in their growth response to TGFβ, with individual clones ranging from strongly growth inhibited to nearly unaffected. These results suggest that multiple factors influence the extent of TGFβ-induced growth effects on both normal and transformed mammary epithelial cells, and that some of these factors may act through epigenetic mechanisms. This work was supported by CA24844 from the National Institutes of Health, Bethesda, MD, and the Office of Energy Research, Office of Health and Environmental Research of the U.S. Department of Energy under contract DE-AC03-76SF00098.     

5′-nucleotidase activity in cultured cell lines. Effect of different assay conditions and correlation with cell proliferation

Summary The 5′-AMPase activity of the ectoenzyme 5′-nucleotidase has been measured in a variety of cell lines, using intact cells. Human cell types showed two orders of magnitude higher enzyme activity than mouse cell lines. The ectoenzyme is inhibited by adenosine 5′-(α,β-methylene) diphosphate and Concanavalin A. A different extent of 5′-nucleotidase lectin inhibition was observed in the studied cell lines, suggesting that the corresponding ectoenzymes are glycoproteins with a different type or degree, or both, of glycosylation. The 5′-nucleotidase activity increased during subculture and decreased after cell transformation. Generally, the 5′-nucleotidase activity was two-to five-fold higher in monolayer than in suspension cell culture. A relation between cell growth and 5′-AMPase activity was also observed. Enzyme activity increased at the end of the lag phase (glioblastoma cells) or during the exponential phase (the other two cell lines). After confluence, the activity decreased to the initial or even lower range of activity. Observed activity variations with cell proliferation correlate with modifications of 5′-AMPase activity during subculture. This work was supported by grant no. PR84-0359 from the Comisión Asesora de Investigación Científica y Técnica (Spain).     

Aberrant in vitro expression of keratin K13 induced by Ca2+ and vitamin A acid in mouse epidermal cell lines.

Normally the expression of the murine type I keratin K13 is restricted to differentiating cells of internal squamous epithelia which line the oral cavity and the upper digestive tract. Recently, however, we were able to show that K13 is aberrantly but constitutively expressed without its normal type II partner K4 also in differentiating parts of 7,12-dimethylbenz(a)anthracene (DMBA/TPA) 12-O-tetradecanoylphorbol-13-acetate-induced squamous cell carcinomas of mouse back skin, whereas its likewise suprabasal expression in papillomas is variable (Nischt et al., Mol. Carcinogenesis 1, 96-108, 1988). In an attempt to reproduce the aberrant expression of K13 in a mouse in vitro system, we have investigated eight established murine epidermal cell lines for their putative ability to express K13. The cell lines differed distinctly in their derivation and comprised cell lines originating from DMBA/TPA induced papillomas (line SP1) or DMBA-treated adult mouse epidermis (line 308) as well as cell lines derived from DMBA or DMBA/TPA-treated primary epidermal keratinocytes (lines PDV and MCA 3D) and cell lines which arose spontaneously by long-term culture of normal epidermal keratinocytes (lines HEL 30 degrees HEL 37 degrees, HELP I and HELP III). We show that, independent of their derivation, all cell lines possess the intrinsic property to aberrantly express K13. Invariably the K13 gene is not expressed when the lines are cultured under low Ca2+ conditions (0.05 mM) and thus prevented from differentiation. Its expression can, however, be induced either by increasing the extracellular Ca2+ concentration or by the addition of physiological concentrations of vitamin A acid to low Ca2+ medium. Whereas in the latter case, K13 expression occurs without concomitant induction of morphological differentiation of the cells, Ca2+ elevation in the culture medium induces squamous differentiation and K13 expression occurs only in differentiating cells, thus reflecting the situation observed in in vivo tumors. All cell lines exhibit a concentration optimum for the stimulatory agents; however, the degree of maximal K13 expression varies considerably among the individual cell lines and shows a striking correlation with the reported tumorigenicity of the lines after transplantation to animals. In contrast, a tentatively suggested correlation between the activation of the Ha-ras gene and the aberrant expression of K13 (Nischt et al., Mol. Carcinogenesis 1, 96-108, 1988) could not definitely be confirmed since we observed K13 expression also in three cell lines which did not carry a mutation in codon 61 of the Ha-ras gene.(ABSTRACT TRUNCATED AT 400 WORDS)     

Analytical characterization of plasma membrane-derived vesicles produced via osmotic and chemical vesiculation

Plasma membrane-derived vesicles are being used in biophysical and biochemical research as a simple, yet native-like model of the cellular membrane. Here we report on the characterization of vesicles produced via two different vesiculation methods from CHO and A431 cell lines. The first method is a recently developed method which utilizes chloride salts to induce osmotic vesiculation. The second is a well established chemical vesiculation method which uses DTT and formaldehyde. We show that both vesiculation methods produce vesicles which contain the lipid species previously reported in the plasma membrane of these cell lines. The two methods lead to small but statistically significant differences in two lipid species only; phosphatidylcholine (PC) and plasmalogen phosphatidylethanolamine (PEp). However, highly significant differences were observed in the degree of incorporation of a membrane receptor and in the degree of retention of soluble cytosolic proteins within the vesicles.     

Differential effects of poly (Glu60, Phe40) (GPhe) on murine TH1 and TH2 cells.

The random amino acid copolymer (Glu60, Phe40)n (GPhe) was previously shown to augment antigen-dependent proliferation of the murine TH2 cell lines DCL-2 and D10.G4.1. In the present study, the addition of GPhe to (Glu36, Lys24, Ala40)n (GLA)-primed BALB/c primary lymph node (1 degree LN) T cell cultures, the source of DCL-2, resulted in significant suppression of both the proliferative and lymphokine response to GLA. Suppression by GPhe of the 1 degree LN response was subsequently shown to be neither antigen- nor haplotype restricted, and was inhibitable by polyclonal anti-GPhe antibodies. Studies were extended to a GLA-reactive T cell hybridoma clone (DL.4G6.1). where significant suppression by GPhe of GLA-stimulated lymphokine production was observed as measured by markedly decreased HT-2 stimulatory activity of the collected supernatants. Subsequent antibody blocking experiments employing the monoclonal anti-murine IL-4 antibody 11B11 revealed that BALB/c GLA-reactive 1 degree LN T cells and DL.4G6.1 did not produce detectable levels of IL-4 in their culture fluids when stimulated by GLA, which suggested that these cells, unlike DCL-2, were TH1-like in nature. The addition of GPhe to the TH1 clones 5.2 and 5.9 resulted in significant suppression of proliferation to homologous antigen (ovalbumin), in contrast to the augmentation observed with the TH2 cell lines DCL-2 and D10.G4.1. It was concluded from these data, that the addition of GPhe to various T cell cultures lead to unusual suppressive and augmenting activities specific for TH1 and TH2 cells, respectively. Although the mechanism for these dichotomous effects of GPhe is as yet undetermined, several possibilities are considered.     

Hypersensitivity and reduced inhibition of DNA synthesis in ataxia telangiectasia lymphoblasts treated with low levels of neocarzinostatin

The effects of neocarzinostatin (NCS) on lymphoblastoid cell lines (LCLs) established from ataxia telangiectasia (A-T) were determined. A-T lymphoblasts were found to be hypersensitive to low levels of NCS as measured by cell growth and cell survival. On the other hand, A-T lymphoblasts failed to postpone DNA synthesis to the same degree as normal lymphoblasts following treatment with NCS. LCLs established from Nijmegen breakage syndrome (NBS) could be distinguished from ataxia and normal cell lines by their intermediate level of survival following exposure to NCS.     

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

Summary We reported that a murine carcinoma (DEN3) and 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.     

The effect of 3T3 fibroblasts on the expression of anchorage independence and cornification of oral keratinocytes

This study examined the effect of 3T3 fibroblasts on the expression of anchorage independence and the degree of cornification in early cultures of three carcinoma-derived epithelial cell lines (R59, R63a, R63b) and in one cell line derived from non-malignant dysplastic epithelium where there was no evidence of invasion (R66a). The epithelial cell lines originated from the palatal (R63a, R66a) and the lingual (R59, R63b) mucosa of rats that had been painted with the carcinogen 4-nitroquinoline N-oxide. In the absence of 3T3 fibroblasts, progressive culture resulted in an increase in the colony forming efficiency (CFE) of R63a, R63b and R59 and a decrease in the percentage of cornified cells in all cell lines. 3T3 fibroblasts caused a decrease in the CFE and the degree of cornification in the 3T3-dependent cell line (R63a), particularly at the lower passages, but these parameters remained essentially unchanged by 3T3 fibroblasts in the 3T3-independent cell lines (R59, R63b). 3T3 fibroblasts did not influence the cornification of R66a and this cell line remained anchorage dependent throughout the study. The results suggest that in malignant cell lines characterised by being independent of 3T3 fibroblasts (R63b, R59) the CFE was inversely correlated to the degree of cornification. However, in the malignant cell line showing a greater dependence on support (R63a) the relationship between CFE and cornification was unclear because these parameters may have been modulated by the presence of 3T3 fibroblasts. The cell line from dysplastic non-invasive tissue (R66a) differed from its malignant counterparts in the fact that CFE and cornification were unaffected by 3T3 fibroblasts despite previous studies showing a dependence on mesenchymal support.     

Histological grading, DNA content, cell proliferation and survival of patients with astroglial tumors

Light microscopy, image cytometry (ICM), and flow cytometry (FCM) were used to study the degree of differentiation, DNA content, and S-phase of astrocytomas and glioblastoma multiforme in 102 patients. The postoperative real survival time (RST) was also studied. Using ICM, 62 astrocytomas were investigated. Grade I astrocytomas were composed of DNA-diploid cell lines, while grade III and glioblastoma multiforme consisted predominantly of DNA-aneuploid lines. Moderately differentiated astrocytomas were divided as follows: 14 DNA-diploid and 18 DNA-aneuploid. Forty astrocytomas were studied by FCM. Using the DNA index (DI) value, cases with abnormal DNA cell lines were established in all astrocytomas, with their number increasing in grades II and III astrocytomas. FCM indicated the same subdivision of moderately differentiated astrocytomas: 12 with DNA-diploid and 12 with DNA-aneuploid stem lines. Patients with DNA-diploid cell lines in the astrocytomas and low S-fraction survived longer than patients with abnormal DNA cell populations and higher S-fraction. The results from this study indicate that, together with the degree of differentiation of astroglial tumors, the appearance of cell lines with abnormal DNA value and higher S-fractions also have prognostic value.     

Modulation of class I and class II histocompatibility antigens on human T cell lines by IFN-gamma

IFN-gamma is an immunomodulatory agent which is known to induce or enhance the expression of class II histocompatibility Ag (Ia Ag) on many lymphoid cells and cell lines of diverse origin. However, we have observed that IFN-gamma did not induce the expression of Ia Ag on Ia- human T cell lines. Neither did IFN-gamma enhance the expression of Ia Ag on Ia+ T cells. However, IFN-gamma was able to enhance the expression of class I histocompatibility Ag (HLA-A,B,C Ag) on a number of the T cell lines tested. Experiments with 125I-labeled IFN-gamma showed a relatively small degree of specific binding to these T cell lines. More extensive studies on two of the T cell lines demonstrated 1000 and 2600 IFN-gamma binding receptor sites/cell and binding affinities of 4.0 X 10(-10) M and 7.3 X 10(-10) M. Thus, although IFN-gamma can bind to human T cell lines and enhance class I histocompatibility Ag on these cells, IFN-gamma alone does not appear to regulate expression of class II histocompatibility Ag on T cell lines.     

Detection of chick rRNA in the cytoplasm of heterokaryons containing reactivated chick red cell nuclei

Collagen fiber formation in vitro was morphologically investigated on 22 epithelial-like cell lines originating from normal rat livers mainly by using specific histochemical stainings. All the cell lines, eight of which were cloned formed typical “collagenous” and/or “reticulin” fibers in the histological sense when they had been left as a full sheet for more than 3 weeks without subculture. Only one of five fibroblastic cell lines serving as control formed fibers to a lesser degree. Different patterns were recognized in fibers appearing between the liver cell cultures and the fibroblast cultures.