Effects of transforming growth factor beta on the anchorage-independent growth of murine epithelial JB6 cells

The work described in this paper demonstrates that transforming growth factor beta (TGF-beta) induces the soft agar growth of murine epidermal JB6 clone 41 (Cl 41) cells. In this regard, TGF-beta is more effective than either 12-O-tetradecanoylphorbol-13-acetate or epidermal growth factor. Together, TGF-beta 1 and epidermal growth factor produce a greater stimulation of soft agar growth than either growth factor alone. In contrast, addition of TGF-beta 1 and 12-O-tetradecanoylphorbol-13-acetate together does not stimulate soft agar growth beyond that produced by TGF-beta 1 alone. Interestingly, retinoic acid inhibits the ability of all three factors to induce the anchorage-independent growth of Cl 40 cells. TGF-beta also exerts long-term effects on Cl 41 cells. This was determined by isolating TGF-beta-induced soft agar colonies and examining their dependence on TGF-beta. Five of the six anchorage-independent clones isolated after TGF-beta 1 treatment were found to exhibit anchorage-independent growth in the absence of TGF-beta. In addition, these clones respond far more strongly to TGF-beta 1 than do the parental Cl 41 cells in terms of both the numbers and the sizes of colonies formed in soft agar. The findings reported here are compatible with the proposal that TGF-beta mediates some effects of 12-O-tetradecanoylphorbol-13-acetate.     

Reversible induction of anchorage independent growth from normal mouse epidermal keratinocytes, MSK-C3H-NU, in soft agar medium by 12-O-tetradecanoylphorbol-13-acetate and epidermal growth factor

The induction of anchorage independent growth occurred in nontumorigenic, mouse epidermal keratinocyte, MSK-C3H-NU cells when inoculated in a 0.3% soft agar medium with 12-O-tetradecanoylphorbol-13-acetate and/or epidermal growth factor. Colonies appeared about 3 weeks after incubation and keratinization of various forms occurred in them. 10-43 cells, a subline of higher subcultivation level, showed a rather sensitive response to these chemicals. 12-O-tetradecanoylphorbol-13-acetate and epidermal growth factor each induced anchorage independent growth but additive effects were also observed. Cell lines were established from clonal growths recovered from these induced colonies in soft agar. Morphologically, they retained their original normal clonal growth and keratinization patterns. Anchorage independent growth was not retained. Response to 12-O-tetradecanoylphorbol-13-acetate and epidermal growth factor, however, became more sensitive in some of the recovered cell lines when compared to their original cells. These data show that the induction of anchorage independent growth in soft agar is reversible in the MSK-C3H-NU cell system.     

Effect of 12-O-tetradecanoylphorbol-13-acetate on two charateristics of transformation acquired sequentially by ENU-exposed rat brain cells.

Cultures derived from rat brains at different times during the latent period of brain-tumour induction by N-ethyl-N-nitrosourea (ENU) showed increased plasminogen activator (PA) activity before being able to form colonies in agar. Control cultures from buffer-exposed animals showed neither property at comparable passages. More detailed investigations, using a culture derived from foetal brains only 2 days after exposure to ENU and clones from this culture, showed a sequence of low PA activity, then increased activity, followed by the ability to form colonies in agar, suggesting progressive transformation of cells in culture. Continuous culturing in the presence of the mouse skin tumour promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), did not accelerate the rate at which these two properties were acquired, but did cause a much greater increase of PA activity once this started to rise. If included in the assay mixture TPA also increased the PA activity of the cells. It therefore appears that in this system TPA can modulate PA activity under certain circumstances.     

Development of an in vitro analogue of initiated mouse epidermis to study tumor promoters and antipromoters

To facilitate the study of skin tumor promotion, a cell culture model system with characteristics analogous to initiated mouse epidermis was established. Cells of the keratinocyte cell line 308, derived from adult mouse skin initiated with 7,12-dimethylbenz[a]anthracene, display the initiated phenotype, since papillomas are produced when the cells are grafted to the backs of athymic mice. Coculture of a small number of these initiated cells with confluent normal primary keratinocytes resulted in the inhibition of growth of colonies of 308 cells. Addition of fresh keratinocytes weekly was required to sustain the inhibition for 3-4 weeks. Inhibition of 308 cell colonies required culture medium with a calcium concentration of 1.2 mM; normal keratinocytes did not inhibit 308 cells in medium with 0.05 mM calcium. Growth of 308 cells was not inhibited by coculture with confluent fibroblasts or by 1.2 mM calcium medium conditioned by either keratinocytes or fibroblasts. During continuous exposure of the cocultures to tumor promoters, 308 cell colonies became apparent within 2-3 weeks. A limited number of promoters were tested in this model system and 12-O-tetradecanoylphorbol-13-acetate, 12-O-retinoylphorbol-13-acetate, mezerein, and benzoyl peroxide were all active. The number of colonies which developed during promoter exposure in cocultures showed a dose-response curve which differed from the dose-response curve for stimulation of growth of 308 colonies in the absence of normal keratinocytes. Simultaneous treatment with 12-O-tetradecanoylphorbol-13-acetate and known inhibitors of skin tumor promotion, such as retinoic acid, fluocinolone acetonide, and bryostatin 1, blocked colony formation of 308 cells in cocultures but not in cultures with only 308 cells. In this model system, the actions of promoters and inhibitors both appear to be mediated by normal keratinocytes.     

Murine leukemia virus-mediated transformation of a mouse epithelial cell line MMC-E

Mouse embryo epithelial cells, MMC-E, were malignantly transformed in culture with MuLV derived from C3H/MCA 5 cells chemically induced by 5-iododeoxyuridine. The frequency of transformation was significantly enhanced if infection was followed by treatment with 100 ng of 12-O-tetradecanoylphorbol-13-acetate per ml. The virus-transformed cells retained their high plating efficiency in soft agar in the absence of 12-O-tetradecanoylphorbol-13-acetate and formed neoplasms in nude mice.     

Effect of 5-azacytidine on differentiation and DNA methylation in human promyelocytic leukemia cells (HL-60)

One of the most readily quantitated indices of myeloid maturation in HL-60 cells is their ability to respond to the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate with increased respiratory burst activity. HL-60 cells exposed to the antileukemic drug, 5-azacytidine (3 to 5 microM) for 24 hr and subsequently cultured in its absence for 2 to 3 days develop an enhanced ability to respond to 12-O-tetradecanoylphorbol-13-acetate with increased respiratory burst activity detectable as an increase both in hexose monophosphate shunt activity and in the proportion of the population producing superoxide anion. 5-Azacytidine treatment also causes marked inhibition of DNA methyltransferase, and thus DNA synthesized by HL-60 cells during the 24-hr period of analogue treatment is essentially devoid of methylated cytosine residues. This suggests, as does our previous finding that a general inhibitor of transmethylation reactions, L-ethionine, can induce differentiation of HL-60 cells, that changes in gene expression triggered by these compounds may be linked to their ability to alter patterns of DNA methylation. Since at least 50% of HL-60 cells capable of forming colonies in soft agar after a 24-hr exposure to 5-azacytidine yield progeny that mature (i.e., produce superoxide anion in response to 12-O-tetradecanoylphorbol-13-acetate) 2 weeks after 5-azacytidine treatment, the results also indicate that the changes induced in HL-60 cells by limited exposure to 5-azacytidine are heritable and can influence gene expression many generations after treatment has been terminated.     

Plant-derived diterpene esters enhance HTLV-I-induced colony formation of lymphocytes in co-culture

The addition to culture dishes of 10-50 ng/ml of the essential diterpene ester of Sapium sebiferum, 12-O-hexadecanoylphorbol-13-acetate (HPA), increased colony formation of normal peripheral blood lymphocytes co-cultured with gamma-irradiated HTLV-I-producing HUT 102 cells. The cells in the stimulated colonies showed an approximately 3-fold increase in the expression of interleukin-2 (IL-2) receptors and a 1.5- to 2.0-fold increase in human T-lymphotropic virus type- I (HTLV-I) p19-positive cells. This biological potency was analogous to that induced by the most potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and stronger than that of 12-O-hexadecanoyl-16-hydroxyphorbol-13-acetate (HHPA) isolated from Aleurites fordii.     

Carba-prostacyclin inhibits 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced transformation in sensitive murine epidermal JB6 cells.

The ability of carba-prostacyclin (cPGI2), a stable analog of prostacyclin (PGI2), to inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced transformation of JB6 cells was investigated. JB6 cells sensitive (P+) and resistant (P-) to TPA-induced transformation to anchorage-independent growth were plated in soft agar in the presence or absence of cPGI2 for 14-21 days. Transformation frequencies were determined by recording colony numbers. cPGI2 was found to inhibit TPA-induced transformation of P+ cells in a dose-dependent fashion with 1 microM cPGI2 producing approximately 50% inhibition of colonies in soft agar. Our findings are consistent with the hypothesis that TPA-induced transformation in JB6 cell variants is mediated by PGI2 via regulation of adenylate cyclase activity and cAMP accumulation, with resultant inhibition of expression of the transformed phenotype, reflected in anchorage-independent growth.     

Defects of cyclic adenosine 3':5'-monophosphate-dependent protein kinases in initiated clones derived from BALB/c 3T3 mouse fibroblasts

Two-stage carcinogenesis is involved in the transformation of mouse fibroblasts BALB/c 3T3 cells. In order to investigate the role of cyclic adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase at the stage of initiation, the following experiments were carried out: (a) two initiated clones (M14, M20) which exhibit 12-O-tetradecanoylphorbol-13-acetate-dependent growth in soft agar medium were isolated from cells treated with N-methyl-N'-nitro-N-nitrosoguanidine. The activity of cAMP-dependent protein kinase in M14 was reduced while that in M20 was similar to the level in parental cells. However, cAMP-binding activity to a regulatory subunit of cAMP-resistant clones were isolated from 4-nitroquinoline oxide- or ethyl methanesulfonate-treated cells. These clones have reduced activities in both cAMP-binding and cAMP-dependent protein kinase itself. Two of three cAMP-resistant clones were proved to be able to grow in soft agar medium only in the presence of 12-O-tetradecanoylphorbol-13-acetate.     

Embryonic mutation as a possible cause of in utero carcinogenesis in mice revealed by postnatal treatment with 12-O-tetradecanoylphorbol-13-acetate

Although in utero irradiation at early stages induced a high incidence of somatic mutations at coat color genes in the embryos of a specified tester strain (PT x HT F1) of mice, it was not carcinogenic by itself. However, in utero-irradiated animals did develop skin tumors and hepatomas (but not leukemias) by the postnatal administration of 12-O-tetradecanoylphorbol-13-acetate. The incidence of both tumors and embryonic mutations increased with in utero doses of X-rays. Furthermore, a large reduction of tumor incidence, about 80%, was observed by low-dose-rate irradiation, similar to the 75% reduction in spot size found for embryonic mutations. The tumor nodule size was also dramatically reduced by low-dose-rate irradiation. Consequently, the induced incidence and size of tumors produced by 12-O-tetradecanoylphorbol-13-acetate treatment parallel those which are observed for coat color mutations as expected, because somatic mutations observed in the pigment cells must similarly occur in embryonic cells of other organs. The larger the clone of mutant cells, the greater their chance of becoming tumorigenic by 12-O-tetradecanoylphorbol-13-acetate posttreatment. These results strongly support the recent epidemiological survey showing that adult types of cancers, but not leukemias, are increasing in the atomic bomb survivors exposed in utero, since humans are continuously exposed to a variety of cancer-promoting agents in contrast to experimental animals reared without such exposures.     

Uniqueness of each spontaneous transformant from a clone of BALB/c 3T3 cells

Seeding 2 X 10(4) cells from a clone of BALB/c 3T3 cells in agar led to the formation of about 100 small colonies (approximate diameter, 0.2 mm) and two large colonies (1-mm diameter). Seven of the former and both of the latter were isolated, and the morphology and growth properties of their cells were observed in repeated weekly passages. The seven subclones derived from the smaller agar colonies spread out on the dish and multiplied more slowly than the parental clone on plastic, but six of them produced more colonies in agar than the parental clone. The two subclones derived from the larger agar colonies had a fully transformed morphology, multiplied much faster than the parental clone on plastic, and produced a high percentage of large colonies in agar. Each of the subclones could be distinguished morphologically from the others and from the parental clone, and most of them could be distinguished on the basis of their colony-forming efficiency in agar. Most of the secondary subclones derived from an early passage on plastic of one of the two large agar clones, died out on the second passage after their isolation. Secondary subclones derived from the same subclone five weeks later had a wide range of fluctuating growth rates, but did not die out on passage. The two rapidly growing subclones derived from large agar colonies initiated fast-growing tumors in nude mice. None of the other subclones produced any visible growth in nude mice over a 3-month period. Large agar colonies of fast growing, morphologically transformed cells appeared once during further passage of the parental clone and two of the subclones. The results reveal a surprising degree of heritable diversity in morphology and growth characteristics of the progeny from a clonal line of nontransformed cells. They also indicate that, in this cell line, only those cells that have a high efficiency (greater than 20%) of large colony formation in agar have the capacity to form tumors in nude mice.     

Altered glucocorticoid binding and action in response to epidermal growth factor in HBL100 cells

Incubation of adherent human breast epithelial HBL100 cells with epidermal growth factor (EGF) decreased [3H]dexamethasone binding by 35% with no effect on affinity. Maximal inhibition was obtained at 3 nM EGF and the 50% effective dose was 0.2 nM EGF. Decreased dexamethasone binding induced by 3 nM EGF was maximal by 5 min of treatment and, in the continuous presence of EGF, persisted at a constant level over 4 days. The action of EGF was antagonized by 12-O-tetradecanoylphorbol-13-acetate, which did not inhibit dexamethasone binding significantly, and by concanavalin A. In homogenates of EGF-treated cells, decreased dexamethasone binding was observed only in the cytosolic fraction. Saturation dexamethasone binding inhibited the growth rate of HBL100 cells by approximately 50%, but concurrent treatment with EGF overcame this inhibition. The effect of EGF on dexamethasone-inhibited cell growth also was antagonized by 12-O-tetradecanoylphorbol-13-acetate.     

Carcinogen-induced phenotypic alterations in mammary epithelial cells accompanying the development of neoplastic transformation

Epithelial cells isolated from the mammary glands of virgin Sprague-Dawley rats and treated with 7,12-dimethylbenz[a] anthracene (DMBA) acquire an indefinite life span and anchorage-independent (AI) growth and form carcinomas in athymic nu/nu mice. Epithelial cells separated from fibroblasts and lipocytes by density-gradient centrifugation after collagenase digestion of the fat pads are grown in a hormone-supplemented medium. Control mammary epithelial cells survived approximately 30 days. After 2 days in culture, the mammary epithelial cells were treated with DMBA (1 microM) for 24 hr allowing for maximum oxidative metabolism of the hydrocarbon. DMBA-treated cells acquired an extended life span and grew in AI medium; however, in most cases, they were nontumorigenic and eventually ceased dividing. A pool of mammary epithelial cells, ME 10CL1, treated with DMBA has grown indefinitely, exhibited AI growth, and after 195 days in culture formed adenocarcinomas when 5 X 10(6) cells were injected into athymic nu/nu mice. When the tumor promoter, 12-O-tetra-decanoylphorbol-13-acetate (100 ng/ml), was added to another pool (ME 11CL2) of DMBA-treated mammary epithelial cells which had been in culture for 110 days, an irreversible increase in cell growth rate and a significant morphological alteration resulted. The 12-O-tetradecanoylphorbol-13-acetate-treated cells also formed colonies in AI medium after 140 days and poorly differentiated carcinomas in athymic nu/nu mice. Inhibition of tumor cell proliferation by tamoxifen is consistent with the mammary origin of the epithelial cells and suggests the presence of a viable estrogen receptor. The results demonstrate in vitro neoplastic transformation of rat mammary epithelial cells by DMBA or promotion of DMBA-initiated cells by 12-O-tetradecanoylphorbol-13-acetate resulting in two different epithelial tumor cell lines.     

Expression of transformed morphology and anchorage independent growth of hamster embryo cells

Formation of morphologically transformed colonies and the abilityto grow in semi-solid agar has been compared for 3 differentcell lines from hamster embryo and for primary hamster embryocells. By manipulating the growth conditions, transformed colonymorphology and growth in agar could be induced for all celltypes studied. Conditions that induced morphologically transformedcolonies, also produced growing colonies in agar. One cell lineand the primary cells needed the presence of the tumor promoter12-O-tetra-decanoyl phorbol-13-acetate for the expression oftransformed morphology and agar growth, while the two othercell lines produced both morphologically transformed coloniesand growth in soft agar without any additions. None of the celllines would produce morphologically transformed colonies inthe presence of newborn bovine serum. Likewise, the cells weredependent on fetal bovine serum in order to grow in soft agar,except for one of the cell lines which produced a low numberof agar growing colonies in newborn bovine serum. The data indicatea close relation between morphological transformation and growthin soft agar.     

Tumor promoter-dependent mouse leukemia cell line

From a spontaneous AKR/Ms thymic leukemia symbiotically cultured with thymic epithelial reticular cells, a tumor promoter-dependent cell line A65T was established by passaging the cells in medium containing 12-O-tetradecanoylphorbol-13-acetate (10 ng/ml). The in vitro growth of A65T was strictly dependent on the presence of active tumor promoters. Their action was reversible, since withdrawal of 12-O-tetradecanoylphorbol-13-acetate resulted in rapid decrease in viability of the cells. Three classes of chemically unrelated compounds sharing tumor-promoting activity in mouse skin could support the in vitro growth of A65T: plant diterpene esters; indole alkaloids; and polyacetates. Their growth effect on A65T cells quantitatively correlated well with the tumor-promoting activity in mouse skin. However, other growth stimulators of epidermal cells such as cholera toxin and epidermal growth factor failed to support the growth of A65T. It is suggested that lymphokines such as interleukin-2 and interleukin-3 were not responsible for 12-O-tetradecanoylphorbol-13-acetate-stimulated growth of A65T because concanavalin A-stimulated spleen cell-conditioned medium containing both interleukin-2 and interleukin-3 activities as well as WEHI-3 cell culture supernatant containing potent interleukin-3 activity did not stimulate the proliferation of A65T cells. Furthermore, 12-O-tetradecanoylphorbol-13-acetate did not induce production of any significant amount of either activity in A65T cells. This cell line is useful for the screening of tumor promoters in environments although, so far, all the compounds capable of stimulating A65T growth have been limited to those competing with phorbol esters for the cellular receptor. Also, the cell line provides a potential model for analyzing growth requirements of developing mouse thymic leukemias.     

Colonies formed in agar from human breast cancer and their identification as T-lymphocytes

Single cell suspensions prepared from human breast cancer specimens by collagenase digestion were cultured in soft agar with phytohemagglutinin-stimulated human lymphocyte-conditioned medium (PHA-LCM). In 6 of 10 different tumors, PHA-LCM-dependent clonal growth was develop. After 12-14 days of incubation, two morphologic types of colony containing 20-500 cells were recognized. Both were composed of lymphocytes of T-cell nature, as judged by cell morphology in smears, cytochemical properties, capacity to form rosettes with sheep erythrocytes, and electron microscopic appearances. Contamination of the tumor cell suspensions by blood could be excluded as a source of the colony-forming lymphocytes, and the incidence of colony-forming cells correlated well with the degree of lymphocyte infiltration of the tumors. Some of the colonies in agar were expanded further in liquid culture in the continuous presence of PHA-LCM. These clones were apparently high in proliferating capacity as compared with the proliferating activity of peripheral T-cell clones obtained from normal blood. These clones were considered to be highly activated T-lymphocytes and to be stimulated to grow in vitro by the T-cell growth factor contained in PHA-LCM. The direct cloning and expansion of such activated T-lymphocytes infiltrating the tumors will be useful for studies on the functional characteristics of these cells.     

Differential sensitivities of transplacentally initiated newborn mouse epidermal cells to different tumor promoters.

Epidermal cells isolated from newborn mice and cultured in low Ca2+ (0.02 mM) medium showed typical basal cell morphology and proliferated as monolayer. An increase in the medium Ca2+ concentration to normal level (1.8 mM) induced terminal differentiation of epidermal cells. Epidermal cells obtained from newborn mice transplacentally initiated with 7,12-dimethylbenz[a]anthracene (DMBA) produced a small number of rapidly growing cellular foci with epidermal morphology when the medium Ca2+ concentration was raised to normal level. The number of these Ca(2+)-induced differentiation-resistant colonies increased with increasing doses of DMBA, indicating that the differentiation-resistant colonies are the cells initiated by DMBA. Three differentiation-resistant colonies were cloned and designated as WY-1, WY-18 and WY-20 cells. All of these cell lines grew rapidly in the normal Ca2+ medium but not in the low Ca2+ medium. A potent skin tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), and other TPA-type second-stage tumor promoters such as mezerein and 12-O-retinoylphorbol-13-acetate, stimulated the growth of WY-18 and WY-20 cells in the low Ca2+ medium, but did not stimulate the growth of WY-1 cells. TPA stimulated DNA synthesis and ornithine decarboxylase induction in WY-18 and WY-20 cells but not in WY-1 cells. A non-TPA-type tumor promoter, okadaic acid, failed to stimulate the growth of these three cell lines. Another non-TPA-type tumor-promoting agent, 7-bromo-methylbenz[a]anthracene, also failed to stimulate the growth of WY-1 and WY-20 cells but stimulated the growth of WY-18 cells. WY-18 and WY-20 cells formed colonies in soft agar but WY-1 cells did not form colonies. When these cell lines were injected s.c. into nude mice, WY-1 cells produced fast-growing tumors, whereas WY-18 and WY-20 cells produced relatively slow-growing tumors. Our present results indicate that each initiated cell has different sensitivities for different types of tumor promoters, and each type of promoter acts on corresponding types of initiated cells.     

Growth of human head and neck squamous cell carcinoma stem cells in agarose

An in vitro human tumor cell assay was used in an attempt to culture head and neck tumors from patients with squamous cell carcinomas. Initially, specimens from nine head and neck tumors were disaggregated by mechanical methods and assayed in soft agar. Five of nine tumors grew in the soft-agar system yielding a cloning success rate of 56%. Plating of 5 X 10(5) cells resulted in 12 to 255 colonies per plate after 21 days in culture, with a cloning efficiency between 0.002% and 0.08%. Recently, the authors replaced the agar with an agarose culture matrix. Of 10 specimens with positive pathology, 9 have shown colony growth (greater than 20 cells). Cloning efficiency in agarose improved approximately 2-fold. Morphologic assessment of tumor colonies in culture showed the same characteristics as those of the original tumor. Overall success rate of growing head and neck tumors in agar and agarose has been 14 of 19 patients (74%). The development of a soft agarose assay for head and neck tumor cells should provide an in vitro technique for predicting in vivo response to anticancer drugs and other therapeutic modalities such as radiotherapy and hyperthermia.