TGF-beta 1 is an autocrine-negative growth regulator of human colon carcinoma FET cells in vivo as revealed by transfection of an antisense expression vector.

Transforming growth factor-beta 1 (TGF-beta 1) has previously been implicated as a potential negative autocrine or paracrine growth regulator of certain cell types (Arteaga, C. L., R. J. Coffey, Jr., T. C. Dugger, C. M. McCutchen, H. L. Moses, and R. M. Lyons. 1990. Cell Growth & Differ. 1:367-374; Hafez, M. M., D. Infante, S. Winawer, and E. Friedman. 1990. Cell Growth & Differ. 1:617-626; Glick, A. B., K. C. Flanders, D. Danielpour, S. H. Yuspa, and M. B. Sporn. 1989. Cell Regulation. 1:87-97). This is based mainly on experiments assessing the effects of exogenous TGF-beta 1 or neutralizing antibodies to TGF-beta 1 on normal or tumor cell proliferation in vitro. However, direct evidence demonstrating such a negative regulation of tumor cell growth in vivo is still lacking. To overcome this problem we have constructed and used an antisense expression vector for TGF-beta 1 as a means of regulating endogenous TGF-beta 1 expression in tumor cells. Antisense-transfected FET human colon carcinoma cells showed a fivefold reduction in TGF-beta 1 mRNA and 15-fold reduction in TGF-beta 1 secretion. Antisense mRNA was detected in transfected cells by an RNase protection assay. Compared to control cells, cultured antisense-transfected cells showed a reduction in lag phase time rather than a change in doubling time. Cloning efficiencies of transfected cells were four times greater than control cells in anchorage-independent assays. Control cells did not form tumors at 5 x 10(5) in athymic nude mice. Antisense-transfected cells formed tumors in 40% of animals injected. At higher inocula (1 x 10(6) cells) antisense-transfected cells formed tumors in 100% of animals injected, but control cells still failed to form tumors. These results show that TGF-beta 1 acts as a negative growth regulator of human colon carcinoma cells in vivo as well as in vitro. Acquisition of partial or full resistance to such inhibitory effects may therefore contribute to tumor development and progression.

Regulation of transforming growth factor alpha expression in a growth factor-independent cell line.

Aberrant transcriptional regulation of transforming growth factor alpha (TGF alpha) appears to be an important contributor to the malignant phenotype and the growth factor independence with which malignancy is frequently associated. However, little is known about the molecular mechanisms responsible for dysregulation of TGF alpha expression in the malignant phenotype. In this paper, we report on TGF alpha promoter regulation in the highly malignant growth factor-independent cell line HCT116. The HCT116 cell line expresses TGF alpha and the epidermal growth factor receptor (EGFR) but is not growth inhibited by antibodies to EGFR or TGF alpha. However, constitutive expression of TGF alpha antisense RNA in the HCT116 cell line resulted in the isolation of clones with markedly reduced TGF alpha mRNA and which were dependent on exogenous growth factors for proliferation. We hypothesized that if TGF alpha autocrine activation is the major stimulator of TGF alpha expression in this cell line, TGF alpha promoter activity should be reduced in the antisense TGF alpha clones in the absence of exogenous growth factor. This was the case. Moreover, transcriptional activation of the TGF alpha promoter was restored in an antisense-TGF alpha-mRNA-expressing clone which had reverted to a growth factor-independent phenotype. Using this model system, we were able to identify a 25-bp element within the TGF alpha promoter which conferred TGF alpha autoregulation to the TGF alpha promoter in the HCT116 cell line. In the TGF alpha-antisense-RNA-expressing clones, this element was activated by exogenous EGF. This 25-bp sequence contained no consensus sequences of known transcription factors so that the TGF alpha or EGF regulatory element within this 25-bp sequence represents a unique element. Further characterization of this 25-bp DNA sequence by deletion analysis revealed that regulation of TGF alpha promoter activity by this sequence is complex, as both repressors and activators bind in this region, but the overall expression of the activators is pivotal in determining the level of response to EGF or TGF alpha stimulation. The specific nuclear proteins binding to this region are also regulated in an autocrine-TGF alpha-dependent fashion and by exogenous EGF in EGF-deprived TGF alpha antisense clone 33. This regulation is identical to that seen in the growth factor-dependent cell line FET, which requires exogenous EGF for optimal growth. Moreover, the time response of the stimulation of trans-acting factor binding by EGF suggests that the effect is directly due to growth factor and not mediated by changes in growth state. We conclude that this element appears to represent the major positive regulator of TGF alpha expression in the growth factor-independent HCT116 cell line and may represent the major site of transcriptional dysregulation of TGF alpha promoter activity in the growth factor-independent phenotype.

Role of Akt2 in regulation of metastasis suppressor 1 expression and colorectal cancer metastasis.

Survival signaling is critical for the metastatic program of cancer cells. The current study investigated the role of Akt survival proteins in colorectal cancer (CRC) metastasis and explored potential mechanisms of Akt-mediated metastasis regulation. Using an orthotopic implantation model in mice, which uniquely recapitulates the entire multistep process of CRC metastasis, combined with an inducible system of short hairpin RNA-mediated Akt isoform knockdown in human CRC cells, our studies confirm a role of Akt2 in CRC cell dissemination to distant organs in vivo. Akt2 deficiency profoundly inhibited the development of liver lesions in mice, whereas Akt1 had no effect under the experimental conditions used in the study. Array analysis of human metastatic genes identified the scaffolding protein metastasis suppressor 1 (MTSS1) as a novel Akt2-regulated gene. Inducible loss of Akt2 in CRC cells robustly upregulated MTSS1 at the messenger RNA and protein level, and the accumulated protein was functionally active as shown by its ability to engage an MTSS1-Src-cortactin inhibitory axis. MTSS1 expression led to a marked reduction in levels of functional cortacin (pcortactin Y421), an actin nucleation-promoting factor that has a crucial role in cancer cell invasion and metastasis. MTSS1 was also shown to mediate suppressive effects of Akt2 deficiency on CRC cell viability, survival, migration and actin polymerization in vitro. The relevance of these findings to human CRC is supported by analysis of The Cancer Genome Atlas (TCGA) and NCBI GEO data sets, which demonstrated inverse changes in expression of Akt2 and MTSS1 during CRC progression. Taken together, the data identify MTSS1 as a new Akt2-regulated gene, and point to suppression of MTSS1 as a key step in the metastasis-promoting effects of Akt2 in CRC cells.

Cell surface proteins and glycoproteins from biologically different human colon carcinoma cell lines.

Six cultured human colon cancer cell lines possessing different biological characteristics were enzymatically radiolabeled in situ with 125I and 3H, and the labeled cell surface proteins and glycoproteins were compared. The electrophoretic patterns of labeled cell surface material suggest correlations between biological properties and cell surface proteins. Highly aggressive cell lines (as assessed by in vitro parameters) had predominant peaks of 125I-labeled proteins between molecular weights 66,000 and 92,500. The major peak of radioiodinated material from the more indolent cell lines occurred between molecular weights 31,000 and 45,000. The profile of one 125I-labeled intermediately aggressive cell line was similar to the profiles of the more aggressive lines, whereas another intermediate line exhibited a profile different from those of both indolent and aggressive lines. Electrophoresis of tritiated material indicated that essentially all of the recovered labeled glycoprotein was of relatively high molecular weight (92,000-180,000) in the indolent lines, whereas the intermediate and highly aggressive lines had patterns with significant peaks between molecular weights 45,000 and 92,500.

Effects of mitomycin on human colon carcinoma cells.

Subpopulations of malignant cells from primary cultures of human colon carcinoma were characterized with respect to their response to mitomycin (MMC). Growth inhibition assays indicated values of 2.06, 0.93, and 0.33 microM for the concentration of drug giving 50% inhibition of growth for sublines HCT 116b, HCT 116, and HCT 116a, respectively. Alkaline elution of filter-bound DNA from cells exposed to MMC in vitro showed a positive correlation between the amount of DNA cross-linking and growth inhibition as a function of drug concentration. Comparable DNA cross-linking was obtained at MMC concentrations of 10 microM for HCT 116b and 5 microM and HCT 116. The cross-linking of DNA from HCT 116a cells at 5 microM MMC was approximately equal to that from HCT 116 cells at doses between 10 and 20 microM MMC. Cross-link removal as a function of time after drug removal of MMC-treated cells was also measured. There was little difference in the rates of alkaline DNA elution after drug removal between HCT 116b and HCT 116a, suggesting that the ability to repair cross-links was not responsible for the differential sensitivities of the cells to MMC. The relative sensitivities of the subpopulations to MMC were reflected in vivo by MMC treatment of nude BALB/c mice bearing xenografts of the cultured sublines.

Enhancement of growth of human colon tumor cell lines by feeder layers of murine fibroblasts.

The effects of feeder layers of C3H/10T 1/2 cells on the growth of human and mouse cell lines were tested. Feeder layers increased colony formation by cultured cancerous cells in semisolid medium over controls grown in semisolid medium without feeder layers. Maximal colony formation was also attained at a faster rate when feeder layers were used. Plating efficiency by cancerous cells obtained directly from xenografts was increased threefold to fivefold in tissue culture when feeder cells were present as confluent monolayers.

Isolation of a cellular subpopulation from a human colonic carcinoma cell line.

A population of cells with increased resistance to 5-fluorouracil was isolated from cultures of the human colonic carcinoma cell line HT29. The resistant cells (HTFU) showed an altered morphology by light and electron microscopy and demonstrated contact inhibition in vitro. DNA assays and chromosome counts showed that HT29 cultures exhibit both hyper- and hypoaneuploidy, while HTFU cultures appear exclusively hypoaneuploid. One year after the isolation of HTFU, both cell lines showed equal sensitivity to 5-fluorodeoxyuridine while HTFU cells retained comparative insensitivity to 5-fluorouracil. Carcinoembryonic antigen production was not demonstrated in pre- or postconfluent cultures of HTFU, although carcinoembryonic antigen was present in both cells and media of HT29 cultures. Growth in semisolid medium was demonstrated for both cultures; however, HTFU showed a lower plating efficiency than did HT29. Tumors were observed in all of the nude mice given injections of HT29 or HTFU cells. Tumors formed from HTFU cells were smaller, and frequently the primary site receded after 6 to 8 weeks; Although in vitro tests suggested a reduced tumorigenic potential for HTFU cells, metastasis was observed only in mice given injections of cells from the HTFU line.

A comparison of methods for the isolation of carcinoembryonic antigen.

Carcinoembryonic antigen (CEA) was isolated from a human tumor with 0.02 M sodium phosphate containing 0.14 M NaCl (pH 7.0) (saline) and further studied after treatment with perchloric acid or 8 M urea. Preparations CEA obtained from saline homogenates and both methods of treatment were characterized by isoelectric focusing and gel filtration. Perchloric acid treatment resulted in an approximate 10-fold decrease in protein and a significant loss of CEA as compared to the saline- and urea-treated material. Isoelectric focusing revealed that the resultant CEA subpopulations were dependent upon the method of isolation. Urea- and saline-treated material showed complex isoelectric patterns that were quantitatively dissimilar. Perchloric acid-treated material showed a comparatively simple isoelectric pattern that was not significantly affected by electrofocusing in the presence of urea. Gel filtration on ACA 34 of the CEA obtained from each method of isolation resulted in two peaks of activity. The first peak corresponded to the void volume of the column, and the second peak coeluted with commercially available purified 125I-labeled CEA. Centrifugation of the peaks obtained resulted in a significantly greater loss of CEA from the void peak of each isolation procedure. The amount of CEA lost from the void peaks following centrifugation differed with each method of isolation and suggested the presence of aggregates.

DNA breakage in human lung carcinoma cells and nuclei that are naturally sensitive or resistant to etoposide and teniposide.

Evidence suggests that the anticancer agents etoposide (VP16-213) and teniposide (VM26) produce DNA breaks and cytotoxicity by interaction with type II topoisomerase. Therefore, levels of type II topoisomerase may influence sensitivity to VP16-213 and VM26. We have characterized four lung carcinoma-derived cell lines for natural sensitivity or resistance to VP16-213 and VM26. Included in this study were two small cell lung carcinoma lines (SW900 and SW1271), an adenocarcinoma line (A549), and a large cell carcinoma (H157). SW1271 was the most sensitive line with a median inhibitory concentration for cell proliferation of 0.5 microM for VM26 and 2.7 microM for VP16-213, and SW900 was the most resistant with median inhibitory concentration values of 2.0 and 16 microM, respectively. A549 and H157 cells were intermediate in sensitivity to these drugs. Alkaline elution techniques were used to study in vivo formation and repair of single and double strand DNA breaks. Single strand DNA breaks were observed in SW1271 cells exposed to as little as 10 nM VM26 or 100 nM VP16-213 for 1 h, whereas SW900 cells required exposure to 10-fold higher concentrations of VM26 or VP16-213 to produce similar results. Single strand DNA breaks predominated only in SW1271 and A549 cells and then, only at low drug concentrations, whereas the ratios between single and double strand DNA breaks decreased at higher drug concentrations. Plots of cytotoxicity versus single and double strand DNA breakage revealed that cytotoxicity produced by both drugs was more closely related to double strand DNA break formation in all four cell lines. DNA breaks appeared rapidly upon addition of drug, reaching plateaus in DNA breaks within 30 min, and repair of both single and double strand DNA breaks occurred rapidly with time to repair one-half of the DNA breaks of 20 to 60 min in all four cell lines upon removal of drug, arguing against repair as a mechanism for drug resistance. DNA breakage was also observed in nuclei isolated from SW900 and SW1271 cells in similar magnitude to that observed in the respective cells. Results indicate that DNA breakage plateaus may reflect a steady-state equilibrium established between the drug and its nuclear target, possibly type II topoisomerase, and suggest that natural resistance to VP16-213 and VM26 may be due to different enzyme levels in sensitive and naturally resistant cells.

N,N-dimethylformamide-induced synthesis of an anti-fibronectin reactive protein in cultured human colon carcinoma cells.

The cell surfaces of human colon cancer cells before and after exposure to N,N-dimethylformamide (DMF) were probed using radioiodination and immunofluorescent labeling techniques. Growth of the human colon carcinoma cell line HCT MOSER in DMF-supplemented culture medium resulted in monolayer culture growth and marked cell morphology alterations consisting of cellular flattening and elongation. Accompanying the morphology alterations were distinct changes in the cell surface protein composition as determined by 125I labeling and electrophoresis. The cell surface changes associated with growth of HCT MOSER cells in the presence of DMF were dependent upon time of exposure to DMF and DMF concentration. Furthermore, removal of DMF-treated HCT MOSER cells from DMF-containing growth medium caused reversion of both cell morphology and cell surface composition to a state comparable to that of cells not exposed to DMF. The HCT MOSER cell surface alterations produced by DMF included a reduction of radioiodinated surface proteins with molecular weights of 87,000, 120,000, and 180,000 and an increase of a 125I-labeled surface protein with a molecular weight of 200,000-250,000. Appearance of a surface protein of approximately 200,000 molecular weight and assumption of a fibroblast-like morphology by DMF-treated HCT MOSER cells suggested that this approximately 200,000 molecular weight material might be fibronectin. Immunofluorescent labeling with anti-human fibronectin showed that HCT MOSER cells grown in DMF did manifest an anti-fibronectin immunoreactive material that was only transiently associated with the cell surface before being released. DMF-treated HCT MOSER cultures continued to express surface carcinoembryonic antigen, indicating that the presence of material immunoreactive with anti-human fibronectin was not secondary to proliferation of a contaminating fibroblast population. The response of HCT MOSER cells to DMF paralleled in many ways that previously reported for methylcholanthrene-transformed AKR-2B (AKR-MCA) fibroblasts. However, unlike AKR-MCA cells, HCT MOSER cells did not exhibit an increase in 125I incorporation per microgram DNA as a function of time of exposure to DMF, which suggests that the surface protein with a molecular weight of approximately 200,000 induced by DMF was not retained on the cell surface.

Occurrence of cytosolic protein and phosphoprotein changes in human colon tumor cells with the development of resistance to mitomycin C.

The cytosolic proteins and phosphoproteins of a mitomycin C-sensitive cell line were examined as progressively greater doses of mitomycin C were administered over a period of 44 weeks. Resistance of the human colon carcinoma cell line increased from a 50% inhibitory concentration of 1 to 6 microM over this time period. Changes in cytosolic protein patterns included increases in the amounts of three proteins with molecular weight X 10(-3)/apparent isoelectric point (Mr/pl) values of 56/6.2, 37/7.3, and 27/6.1. Analysis of in vitro 32P-labeled phosphoprotein patterns revealed reductions in the amounts of four proteins with Mr/pl values of 42/6.3, 40/6.7, 31/6.3, and 25/6.1. One increase was detected in a phosphoprotein with a Mr/pl value of 33/6.1. These changes in cytosolic components paralleled the development of resistance to mitomycin C and may reflect changes in the clonal composition of the cell line as it becomes progressively more resistant to mitomycin C or changes in critical proteins or enzymes involved in the activation or biotransformation of the drug.

Cell surface features associated with differentiation-induction of methylcholanthrene-transformed AKR-2B fibroblasts by N,N-dimethylformamide.

Methylcholanthrene-transformed AKR-2B mouse embryonal fibroblasts (AKR-MCA cells) were examined for cell surface alterations after growth in culture medium containing N,N-dimethylformamide (DMF) using the lactoperoxidase-glucose oxidase radioiodination procedure with subsequent electrophoresis. DMF has been shown to induce maturational changes in a variety of transformed cells in vitro and has been reported to produce a more normal phenotype when applied to cultured AKR-MCA cells. The electrophoretic profile of 125I-labeled surface proteins from AKR-MCA cells exhibited a prominent peak of labeled material with a molecular weight of approximately 85,000. After growth of AKR-MCA cells in medium containing DMF, the Mr 85,000 peak was substantially reduced, while there was a large increase in Mr 200,000 to 250,000 radioiodinated surface material. This cell surface labeling pattern was virtually identical to that of the nontransformed AKR-2B fibroblasts from which AKR-MCA cells were derived. The cell surface alterations observed upon exposure of AKR-MCA cells to DMF occurred as a function of time of growth in DMF and DMF concentration. Growth of AKR-MCA cells in DMF resulted in a steady increase in cell surface 125I incorporation up to the fourth day of exposure to DMF. At this time, the incorporation level was 22.9-fold greater than that for untreated AKR-MCA cells. Incorporation of radiolabel was decreased after the fifth and sixth days of AKR-MCA exposure to DMF. This trend was also manifested by AKR-2B fibroblasts grown in the presence of DMF. The data suggest that there was increased expression of the Mr 200,000 to 250,000 surface protein on both AKR-2B and AKR-MCA cells when grown in DMF. DMF also inhibited morphological transformation and the cell surface changes associated with transformation of AKR-2B cells by exogenous transforming growth factors.

beta-Hexosaminidase isozymes in human colonic carcinoma.

Isoelectric focusing of crude extracts demonstrated that human colonic carcinomas contained a higher proportion of beta-hexosaminidase B than beta-hexosaminidase A, while normal human colonic mucosa contained a higher proportion of the A form of the enzyme. Studies of the isolated isozymes showed that beta-hexosaminidase B was more stable to heat and more active at low pH than beta-hexosaminidase A. Kinetic studies revealed that the A and B forms of beta-hexosaminidase had essentially the same Km and Vmax for p-nitrophenyl-N-acetyl-beta-D-glucosaminide.

Effects of difluoromethylornithine and dicyclohexylammonium sulfate on the transformed state of AKR-MCA cells.

The effect of two inhibitors of polyamine biosynthesis, difluoromethylornithine and dicyclohexylammonium sulfate, on the transformed fibroblastic cell line AKR-MCA and its parental counterpart AKR-2B was investigated. Treatment of monolayer AKR-MCA cells with either agent results in morphological changes akin to AKR-2B; the cells appear to be flattened with a polygonal shape. The ability of the inhibitors to alter the phenotype is lost when the cells are cocultured with polyamines. More specifically, putrescine and spermidine abrogate the effects of difluoromethylornithine while only spermidine is effective in reversing the dicyclohexylammonium sulfate induced phenomenon. Further evidence that these enzyme inhibitors are reversing the transformed state of AKR-MCA is obtained from soft agarose experiments. AKR-MCA cells will generate colonies only in the absence of either difluoromethylornithine or dicyclohexylammonium sulfate. Polyamine levels were determined in parental AKR-2B and AKR-MCA cells. The levels of putrescine and spermine were similar in both cell types. In contrast, significantly more (P less than or equal to 0.01) spermidine was expressed by the malignant line [7.3 +/- 0.8 (SD) nmol/10(6) cells] when compared with the untransformed AKR-2B (5.4 +/- 0.8 nmol/10(6)) cells. Intracellular putrescine and spermidine were sensitive to difluoromethylornithine, dicyclohexylammonium sulfate, and dimethylformamide, a planar, polar solvent which has been reported to "normalize" the transformed phenotype. AKR-MCA treated with difluoromethylornithine or dimethylformamide manifested time dependent reductions in both polyamines which preceded morphological changes. Dicyclohexylammonium sulfate similarly caused a 70% reduction in spermidine, but in contrast to the other agents there was a marked accumulation of putrescine. These data concur with the established molecular actions of the two enzyme inhibitors as blockers of ornithine decarboxylase (difluoromethylornithine) and spermidine synthase (dicyclohexylammonium sulfate). The normalizing capacity of dimethylformamide was not compromised by cotreatment with putrescine or spermidine. Both difluoromethylornithine and dicyclohexylammonium sulfate inhibited the growth of monolayer AKR-2B and AKR-MCA. In view of the well documented cytostatic effects of polyamine inhibitors, it is suggested that a decrease in growth by these agents triggers a more normal phenotype in AKR-MCA cells.

Induction of carcinoembryonic antigen secretion and modulation of protein secretion/expression and fibronectin/laminin expression in human colon carcinoma cells by transforming growth factor-beta.

We have recently reported that TGF-beta induces a response similar to that of planar polar differentiation promoters in human colon carcinoma MOSER cells. N,N-Dimethylformamide and TGF-beta had similar effects on MOSER cells with respect to reversible inhibition of growth (both in monolayer culture and semisolid medium), induction of fibronectin expression and the induction of morphological alterations (Cancer Res., 47:2950-2954, 1987). Since the expression of carcinoembryonic antigen (CEA) has been reported to be modulated by planar polar compounds that promote differentiation in colon carcinomas, we addressed the issue of whether the differentiation-like effects of TGF-beta on these cells would also encompass modulation of CEA expression in the MOSER cells. The biological modulating effects of TGF-beta on extracellular matrix glycoprotein expression and the expression and secretion of cellular proteins were also studied in view of the reported modulating effects of this growth factor on untransformed, noncolonic cells. In this communication we report that TGF-beta induced the synthesis of fibronectin and laminin but not collagen IV. TGF-beta also induced CEA secretion in a dose-dependent manner. Elevated CEA secretion was detected following 48 h of TGF-beta treatment and a 16-fold increase in CEA secretion was observed following 7 days of treatment. The cells were committed to secrete CEA following one dose of TGF-beta treatment. The enhanced expression of four cellular proteins (Mr 42,000, Mr 48,000, Mr 52,000, and Mr 55,000) and the enhanced secretion of three proteins (Mr 66,000, Mr 200,000, and Mr 400,000) were also induced. Some of these protein alterations were detected as early as 6-24 h following TGF-beta treatment. It is concluded that TGF-beta modulated the production and secretion of CEA, the synthesis of fibronectin and laminin, and the expression and secretion of several cellular proteins in the colon carcinoma MOSER cells. To our knowledge, this is the first report on the modulation of CEA and laminin by TGF-beta in tissue-cultured cells, and is the first report on the modulation of cellular proteins by this growth factor in human colon carcinoma cells.

Rate of growth and extent of differentiation reflected by cytoplasmic proteins and antigens of human colon tumor cell lines.

The cytosolic proteins and antigens of 11 human colon tumor cell lines were examined with respect to their rate of growth and state of differentiation. Coomassie blue-stained protein analysis of sodium dodecyl sulfate-containing polyacrylamide gels revealed protein bands at Mr 30,000, 31,000, and 58,000, which were characteristic of slower growing and more differentiated cell lines. More rapidly dividing and disdifferentiated colon cell lines lacked the Mr 30,000 and Mr 58,000 bands; instead, they produced a single protein band that ran between the Mr 30,000 and Mr 31,000 positions on the gel. Western transfer analysis of cytoplasmic antigens further subdivided the 11 cell lines into 3 separate categories. Slowly growing and more differentiated lines produced a Mr 52,000 antigen. Intermediate lines, with respect to growth rate and state of differentiation, produced a Mr 38,000 antigen. The rapidly growing and highly disdifferentiated cell lines contained three cytosolic antigens with molecular weights of 37,000, 39,000, and 48,000. These criteria made it possible to classify these 11 human colon tumor tissue culture cell lines into 3 groups which reflect their state of growth activity and degree of differentiation.

Changes in receptor occupancy and growth factor responsiveness induced by treatment of a transformed mouse embryo cell line with N,N-dimethylformamide.

Treatment of the transformed mouse embryo fibroblast cell line (AKR-MCA) with N,N-dimethylformamide (DMF) results in a reversion to the nontransformed AKR-2B cell line phenotype. AKR-MCA cells grown in the presence of 1% DMF showed a 2-fold increase in the sites for epidermal growth factor (EGF) binding. However, most of these sites were occupied by an endogenous ligand. The EGF receptor was unoccupied in untreated AKR-MCA cells. The increased receptor occupation was paralleled by an increase in the mitogenic response to EGF. Treatment of these cells with 1% DMF resulted in a 6-fold stimulation of mitogenesis by EGF. The ability to respond to nutrient replenishment (a property of growth-arrested AKR-MCA cells) was lost within 24 h of DMF treatment. Upon removal of DMF from the cells, both the mitogenic response to EGF and the occupation of the EGF receptor by endogenous ligands were lost. Treatment of the AKR-2B cell line with DMF had little effect on its growth properties. Therefore, DMF altered the growth control response and growth factor binding of AKR-MCA cells in a reversible, noncytotoxic manner.

Single- and double-strand DNA breakage and repair in human lung adenocarcinoma cells exposed to etoposide and teniposide.

The anticancer agents 4'-demethylepipodophyllotoxin-4-(4,6-O-ethylidene-beta-D-glucopyra noside (etoposide) (VP16-213) and 4'-demethylepipodophyllotoxin-4-(4,6-O-thenylidene-beta-D-gl ucopyranoside (teniposide) (VM26) produce cytotoxicity by inhibiting type II topoisomerase, resulting in an accumulation of DNA breaks. By using alkaline elution techniques to assess in vivo DNA break frequencies, we have been able to follow formation and repair of both single- and double-strand DNA breaks induced by the exposure of A549 human lung adenocarcinoma cells to VP16-213 and VM26. Single-strand DNA breaks are detectable in cells within 2 min of drug exposure, increase in frequency to a maximum after as little as 15 min of exposure, and remain near maximum levels. Double-strand breaks accumulate more slowly, reaching a maximum after 1 to 2 h, and remaining constant thereafter upon continuous exposure to drug. Single-strand DNA breaks predominate at early incubation times and low drug concentrations, whereas the ratios between single- and double-strand DNA breaks decrease at higher drug concentrations. Changing to drug-free medium after 1-h drug exposure results in rapid exponential repair of both single- and double-strand DNA breaks with a time required for repair of one-half of the DNA breaks of 20 to 60 min. VM26 and VP16-213 have similar kinetics for DNA break formation and repair and similar relationships between DNA breakage and cytotoxicity, but VM26 is five to ten times more potent than VP16-213. Results indicate that DNA breakage plateaus may reflect a steady state equilibrium established between the drug and its nuclear target, possibly type II topoisomerase, and demonstrate unique properties of VP16-213- and VM26-induced DNA breakage.

Isoenzymes of beta-hexosaminidase from normal rat colon and colonic carcinoma.

N-Acetyl-beta-D-hexosaminidase isolated from normal rat colon was compared to that obtained from a transplantable rat colonic carcinoma. Levels of total hexosaminidase from purified epithelial cells of normal colon were similar to those from purified malignant cells from the transplantable tumor. Cultured malignant cells had significantly higher levels of activity than did freshly purified tumor cells. Isoelectric focusing of hexosaminidase from normal rat colon indicated approximately equal amounts of A (pI 5.0 ) and B (pI 8.1) isoenzyme activity. The B isoenzyme (normal cell) was more stable to heat inactivation than was the A isoenzyme and had significantly higher activity at low pH's. In contrast, the B isoenzyme from the tumor was relatively unstable to heat and low pH.

Reversion of transcriptional repression of Sp1 by 5 aza-2' deoxycytidine restores TGF-beta type II receptor expression in the pancreatic cancer cell line MIA PaCa-2.

The pancreatic cancer cell line, MIA PaCa-2 is not responsive to transforming growth factor beta (TGF-beta) because of a lack of expression of the TGF-beta type II receptor (RII). We show that the lack of RII expression is caused by a deficit of the transcription factor Sp1. Nuclear run-off assays and Western immunoblot showed low levels of transcription and protein levels of Sp1, respectively. Treatment of MIA PaCa-2 cells with the DNA methyl transferase inhibitor, 5-aza-2'-deoxycytidine, resulted in an increase in the rate of Sp1 transcription, in Sp1 protein expression, and in the binding of Sp1 to the RII promoter. Ectopic expression of Sp1 cDNA in MIA PaCa-2 cells led to an increase in RII promoter-chloramphenicol acetyltransferase activity and RII expression. Expression of Sp1 cDNA also caused a reduction in both growth and clonogenicity that was associated with restoration of responsiveness to TGF-beta. Conversely, cells that express RII (BxPC-3 and MIA PaCa-2 Sp1 transfectants) when treated with mithramycin, an inhibitor of Sp1 binding, showed a reduction in RII mRNA expression. The reduction of RII mRNA was attributed to a decrease in RII promoter-chloramphenicol acetyltransferase activity that was associated with a decrease in Sp1 binding to the RII promoter. These data indicate that transcriptional repression of the Sp1 gene in MIA PaCa-2 cells plays a role in the transcriptional inactivation of the RII gene and thus lack of responsiveness to TGF-beta.