Suppression of keratinocyte differentiation in SSC-9 human squamous carcinoma cells by benzo[a]pyrene, 12-O-tetradecanoylphorbol-13-acetate and hydroxyurea

In the human squamous carcinoma cell line SCC-9, the expression of two markers of keratinocyte differentiation, involucrin and transglutaminase, was greatly stimulated when growing cultures reached confluence. However, the two markers differed temporally in their induction, with transglutaminase reaching maximal levels shortly after confluence and involucrin a week later. If replication was arrested with hydroxyurea prior to confluence, transglutaminase induction occurred within several days but involucrin levels were completely suppressed. Such a striking degree of uncoupling also resulted when the cells were treated with polycyclic aromatic hydrocarbons such as benzo[a]pyrene but not with 2,3,7,8-tetrachlorodibenzo-p-dioxin, a potent inducer of aryl hydrocarbon hydroxylase, or with pyrene. Chronic treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate suppressed expression of both transglutaminase and involucrin. However, suppression of the latter (evident in greatly reduced mRNA levels) was considerably more potent and powerful. These findings demonstrate uncoupling of keratinocyte differentiation, potentially useful in analysis of its multiple regulatory influences. They also emphasize the utility of sensitive keratinocyte targets for studying the mechanisms by which model carcinogens disturb the orderly progression of events in their differentiation program.     

Phenotypic changes of human neuroblastoma cells in culture induced by 12-O-tetradecanoyl-phorbol-13-acetate

SK-N-SH and SH-SYSY human neuroblastoma cells treated by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) express morphological and biochemical changes, which indicate that differentiation towards more mature cells has occurred. The most prominent morphological changes were the development in 40–60% of the cells of cell-surface projections longer than 50 μm and cytoplasmic neurosecretory granules demonstrated by electron microscopy. At the biochemical level, TPA induced a two-fold increase in the relative activity of neuron-specific enolase and 30- to 40-fold increase in noradrenaline and adrenaline concentrations. A decrease in proliferation rate of TPA-treated cells was observed. The biological effects of TPA were slightly potentiated by nerve growth factor.     

Differing patterns of cytotoxicity of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate in various human cell strains

Inhibition of colony formation by the phorbol ester tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) over a wide range of concentrations (10(-4)-10(2) ng/ml) was examined in two normal human diploid fibroblast strains and eight cell lines derived from various human tumors. Three dose-response patterns were observed: (i) no killing at any dose, which is characteristic of rodent cells; (ii) increasing cytotoxicity with TPA doses of 0.1 ng/ml or greater; and (iii) a biphasic response with maximal cytotoxicity at 1.0 ng/ml, and minimal effects at much lower or higher concentrations. The latter response group included both normal and tumor cell strains. When normal cells were incubated concurrently with superoxide dismutase or CuDIPS, survival was enhanced in a dose-dependent manner. Specific binding of [3H]PDBu to cells from each of the three response categories was studied to determine whether the cells might contain two classes of specific phorbol ester receptors. Scatchard plots yielded straight lines, consistent with one class of binding sites. The possible significance of this cytotoxic effect of TPA in human cells at dose levels usually considered typical for specific phorbol ester responses is discussed.     

Differential regulation of vimentin mRNA by 12-O-tetradecanoylphorbol 13-acetate and all-trans-retinoic acid correlates with motility of Hep 3B human hepatocellular carcinoma cells

Vimentin is a growth-related gene and often expressed when epithelial cells are stimulated to proliferate by growth factors. In cancer, vimentin expression is associated with a dedifferentiated malignant phenotype, increased motility, invasive ability and poor prognosis. We studied the regulation of vimentin mRNA and multistep invasion processes following treatment of 12-O-tetradecanoylphorbol 13-acetate (TPA) and all-trans-retinoic acid (RA) in Hep 3B hepatocellular carcinoma cells. TPA showed marked induction of vimentin mRNA, while RA decreased the mRNA level. TPA or RA did not affect cell proliferation, cell-matrix protein adhesion, and matrix metalloproteinases and urokinase plasminogen activator activities. In vitro invasion ability was significantly increased or decreased with TPA or RA treatment, paralleled to the in vitro motile activity, respectively. These findings suggest that TPA and RA could modulate the invasive potential of Hep 3B cells by altering cellular motility related to differential regulation of vimentin mRNA.     

Molecular Mechanism of Differentiation and Apoptosis of U937 Cells Induced by 12-O-Tetradecanoylphorbol-13-Acetate

OBJECTIVE To explore the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on differentiation, apoptosis and related molecular mechanisms in U937 myelomonocytic leukemia cells.METHODS Morphological changes were analyzed by phase contrast and light microscopy, expression of the monocytic differentiation maker CD11b by direct immunofluorescence staining, cell cycle distribution and apoptosis by flow cytometry, and expression of bcl-2, Bax, survivin and p21Cip1/Waf1proteins by Western analysis.RESULTS Treatment of U937 cells with 10 nmol/L TPA induced cell adherence. The adherent cells showed G0/G1 cell cycle arrest (69.0% at 24h vs 52.1% control; P< 0.01),and morphologic changes and increased expression of the monocytic differentiation marker CD11b (63.0% at 72 h vs15.3% control; P< 0.01 ). In addition to these effects, about 20% of the cells still remained in suspension and exhibited a time-dependent increasing apoptosis, which reached 70.3% after 72 h of treatment ( P< 0.01 ). TPA treatment for 24 h induced expression of p21Cip1/Waf1 in the adherent cells, but not in the non-adherent cells. Furthermore, bcl-2 and survivin expression declined in 24 h-TPA-treated non-adherent cells compared with untreated control and adherent cells, whereas no change in the expression of Bax was detected.CONCLUSION TPA induces both differentiation and apoptosis in U937 cells,which may be related to the upregulation of p21Cip1/Waf1 and downregulation of bcl-2 and survivin expression.     

Response of Walker 256 carcinosarcoma cells to 12-O-tetradecanoylphorbol 13-acetate: possible regulation by endogenous cyclooxygenase metabolites

Walker 256 carcinosarcoma cells (Walker cells) maintained in suspension culture responded to stimulation with 12-O-tetradecanoylphorbol 13-acetate [(TPA) CAS: 16561-29-8] by becoming temporarily adherent to the substratum. Both the control and treated cells produced very low levels of cyclooxygenase metabolites as detected by radioimmunoassay procedures. Levels of prostaglandin F2 alpha, 6-keto prostaglandin F1 alpha (6-keto PGF1 alpha) (a prostacyclin metabolite), and thromboxane B2 were virtually the same as background, and prostaglandin E2 (PGE2) levels were only slightly higher. Studies employing high-performance liquid chromatography also failed to detect significant quantities of cyclooxygenase products in the supernatants from either the control or the stimulated Walker cells. Although the Walker cells maintained in culture failed to produce significant amounts of cyclooxygenase metabolites, they produced much greater amounts of these products, particularly PGE2 and 6-keto PGF1 alpha when they were maintained as an ascites tumor. Concomitant with the production of these metabolites was a loss in responsiveness to TPA in the adherence assay. Upon reestablishment in culture, the cells gradually reacquired the ability to respond to TPA. Over the same period, synthesis of cyclooxygenase products was curtailed. If the cells taken from ascites tumors were incubated with indomethacin so as to inhibit the production of cyclooxygenase metabolites, they rapidly regained responsiveness to TPA. These findings suggest that stimulus-coupled responses in the Walker cells may be regulated, at least in part, through the production of endogenous cyclooxygenase metabolites.     

Tumor promoter 12-O-tetradecanoylphorbol 13-acetate: effect on complement and Epstein-Barr virus receptors in human lymphoblastoid cell lines

The effect of the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) on the C3 and Epstein-Barr virus (EBV) receptors in various human lymphoblastoid cells was investigated with the use of the erythrocyte-antibody-complement (EAC) rosette formation method and a quantitative bioassay for EBV receptors. TPA caused a significant decrease of C3 receptors in the cultures of both Raji and SB4 cells (by approximately 50% of the C3 receptors in untreated cultures at 10 ng/ml). Kinetic studies revealed that the rate of reduction was rather moderate but progressive, reaching a maximum 5 days after treatment with TPA. Kinetic studies showed that EBV receptors also decreased, similar to the reduction seen with C3 receptors after TPA treatment. The effect of TPA on the reduction of C3 receptors was observed not only in these cells but also in other EBV-positive B-cells, subclones of SB4 cells, and MOLT-4 cells. However, in an EBV-negative B-cell line, BJAB, EAC rosette formation was significantly enhanced.     

Retinoic acid suppression of squamous differentiation in human head-and-neck squamous carcinoma cells.

Retinoids (vitamin A analogues) inhibit the squamous differentiation of normal and malignant epithelial cells. This study investigated the ability of the head-and-neck squamous-cell carcinoma (HNSCC) cell line 1483 to undergo squamous differentiation in the absence and presence of beta-all-trans retinoic acid (RA). The growth of these cells in culture is accompanied by an increase in keratinocyte transglutaminase, involucrin and keratin KI, 3 established markers of squamous cell differentiation. Higher levels of these differentiation markers were detected in cells cultured in delipidized serum (DLS), from which endogenous retinoids have been extracted, than in cells cultured in fetal bovine serum (FBS), which contains retinoids. Treatment with I microM RA decreased the levels of the various differentiation markers in cells cultured in either FBS or DLS as revealed by immunofluorescent labelling of permeabilized cells and by immunoblotting of cell extracts using specific monoclonal or polyclonal antibodies. The cells' ability to cross-link proteins to form envelopes under the plasma membrane was stimulated in the presence of calcium ionophore but inhibited by RA. These results indicate that the malignant 1,483 HNSCC cells recapitulate the main characteristics of normal squamous-cell differentiation in culture and that RA suppresses this differentiation as it does in normal keratinizing epithelial cells.     

Differentiation-associated changes in human non-T, non-B leukemia cell lines after treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA)

The ability of TPA to induce stable phenotypic changes that normally serve as markers of differentiation was examined in the four human non-T, non-B cell lines, NALL-1, NALM-16, REH and KM-3. In all four lines, noncytotoxic concentrations of the phorbol ester caused an extensive reduction in the number of cells expressing cALL surface antigen and terminal deoxynucleotidyl transferase. The disappearance of these markers correlated with the loss of cell proliferation. In one of the cell lines, NALL-1, TPA treatment gave rise to a significant increase in Ia-like antigen and antigen T-101, markers which represent more advanced stages of cell maturation. However, surface or cytoplasmic immunoglobins, indicators of mature B cells, were not detectable. Antigen 3A1, specific for myeloid and for T cells, antigen Leu-4, specific for T cells and antigen CM1, specific for monocytes, were also absent. In all cell lines, exposure to TPA resulted in an approximately two-fold increase in acid phosphatase and beta-glucuronidase activity. The emergence of these phenotype changes was not altered upon repeated washing of the TPA-treated cells. These results demonstrate that while TPA is capable of inducing various non-T, non-B cell lines to differentiate to a limited degree, differences exist between the lines in the extent to which they can mature towards the B-cell stage.     

Growth inhibition of human melanoma-derived cells by 12-O-tetradecanoyl phorbol 13-acetate

In vitro growth of 6 human melanoma-derived cell lines was inhibited markedly by the phorbol-ester tumor promoter 12-O-tetradecanoyl phorbol 13-acetate (TPA), a potent activator of several isoforms of protein kinase C (PKC). Utilizing PKC isoform-specific antibodies in immunoblotting experiments, we found that the PKCα and PKC isoforms were expressed in all of the 6 melanoma cell lines tested, whereas the PKCβ isoform was expressed at detectable levels in only 2 of the 6 cell lines. The SK-Mel-173 melanoma cell line, which had relatively high levels of PKCβ mRNA and protein expression, and which was also the most sensitive to cell growth inhibition by TPA, was used to isolate clones whose growth was less inhibited by TPA. Immunoblotting experiments revealed that in parental SK-Mel 173 cells PKCβ was rapidly down-regulated to below detectable levels after treatment for 48 hr with TPA, but that in TPA-resistant variant clones there was negligible down-regulation of PKCβ by TPA. On the other hand, treatment of parental and TPA-resistant SK-Mel 173 cells with TPA led to partial down-regulation of PKCα in both cell lines. Total PKC enzyme activity was also greater in TPA-resistant cells than in parental SK-Mel 173 cells. Our results show that TPA might inhibit the growth of melanoma cells by causing down-regulation of specific isoforms of PKC that are required to maintain the growth of these cells.     

Glycosaminoglycan synthesis during differentiation of HL60/HGPRT-leukemia cells induced by dimethyl sulfoxide and 12-O-tetradecanoylphorbol-13-acetate

Glycosaminoglycans (GAGs) are polyanionic components of the cell surface that have been shown to play an important role in the cellular differentiation of many embryonic systems, as well as in the maturation of the developing human leukocyte. For this reason, the production of GAGs during the induction of myelocytic and macrophage-like differentiation of the human promyelocytic leukemia cloned cell line HL60/HGPRT- was studied. The major GAG component of HL60/HGPRT- was chondroitin 4-sulfate. This molecule has been reported to be the major GAG constituent of normal granulocytes and myeloid leukemia cells as well. Treatment of HL60/HGPRT- cultures with dimethyl sulfoxide, which initiates myeloid maturation, or 12-O-tetradecanoylphorbol-13- acetate, which induces the formation of macrophage-like cells, resulted in a 43 and 34% reduction, respectively, of the incorporation of [35S]sulfate into total GAGs at a time when greater than 80% of the cells were morphologically immature and were unable to reduce nitroblue tetrazolium dye. This reduction occurred primarily in GAGs associated with the cells, which decreased by 75% after exposure to these agents. Therefore, the distribution of GAGs between the cellular and medium compartments was altered by exposure to inducers. A phorbol ester with no capacity to induce differentiation, 4 alpha-phorbol-12, 13-didecanoate, elicited a reproducible but less dramatic decrease in cell-associated GAGs. The reduction in [35S]-sulfate incorporation into GAGs, therefore, may be an important step in leukocyte differentiation and may provide a useful biochemical probe of the maturation process.     

Tumor promoter 12-O-tetradecanoylphorbol-13-acetate receptors in normal human transitional epithelial cells

As a prelude to study the promotion with TPA of in vitro transformationof human urothelial cells (HUC) in culture, we characterizedtumor promoter TPA receptors in primary cultures of HUC. [³H]TPAbound specifically to intact living HUC; maximum specific bindingwas attained in 30 min at 37°C. [³H]TPA bound to HUC ina saturable and competitive manner. Scatchard analysis of specificbinding to intact cells displayed a single slope correspondingto an equilibrium dissociation constant (Kd) of 0.56 nM; atsaturation TPA-binding capacity was 2.37 pmol/10⁶ HUC (1.43x 10⁶ sites per cell). [³H]TPA bound specifically and with highaffinity to the particulate fractions of HUC; binding was bothsaturable and reversible. Saturation of the specific bindingof [3H]TPA occurred at 1 nM at 4°C. Scatchard analysis ofspecific binding to the particulate fraction displayed a singleslope corresponding to a Kd of 1.08 nM; at saturation TPA-bindingcapacity was 2.05 pmol/mg protein (750 000 molecules per HUC).[³H]TPA binding was inhibited by the biologically active phorbolester, phorbol didecanoate, whereas inactive phorbol did notcompete for TPA binding. Binding was not affected by sodiumsaccharin, epidermal growth factor, retinoic acid or dexamethasone.[³H]TPA bound specifically to the HUC cytosolic fraction butonly in the presence of calcium and phosphatidylserine. Calcium-activatedand phospholipid-sensitive protein kinase activity was detectedin HUC fractions. These results indicate the presence of high-affinityspecific receptors for TPA in HUC.     

Phorbol 12-myristate 13-acetate (pma) induces neuroendocrine-like differentiation and reverses Doxorubicin-resistance of human colon-carcinoma cells in-vitro

Human colon carcinoma LoVo/DX cells, which have been selected from parental LoVo for resistance to doxorubicin, express a typical multidrug resistant (MDR-1) phenotype. We have investigated whether phorbol 12-myristate 13-acetate (PMA) which often induces phenotypical changes in human tumor cells could, at the same time, modulate differentiation and sensitivity of LoVo/DX cells to doxorubicin. After 48 h exposure to 100 nM PMA, morphological changes became evident on LoVo/DX cells which showed elongated cytoplasm and dendritic-like structures: moreover immunocytochemical findings were suggestive of neuroendocrine-like differentiation. Under the same experimental conditions, LoVo/DX became sensitive to doxorubicin and showed enhanced intracellular drug-accumulation and reduced membrane expression of the 170 kD glycoprotein GP-170, which is the cellular product of the mdr1 gene. We conclude that pharmacological induction of tumor cell differentiation by PMA is paralleled by abrogation of drug resistance in a colon carcinoma MDR-1 cell line.     

Interleukin 13 participates in terminal differentiation of esophageal squamous cell carcinoma cells

BackgroundIn China, esophageal squamous cell carcinoma (ESCC) accounts for more than 90% of all esophageal cancer cases. Interleukin 13 (IL-13) was widely reported to play a key role in tumor progression. Our previous study reported that IL-13 was a favorable predictive marker for the overall survival of esophageal squamous cell carcinoma (ESCC) patients, but how IL-13 contributes to ESCC progression remains unknown. This study aims to explore the role of IL-13 and its underlying downstream molecular mechanisms in ESCC progression.MethodsTissue microarrays including 262 primary ESCC tumor tissues were collected and analyzed. The expression of IL-13 in ESCC tumor tissue was detected with immunohistochemistry staining (IHC). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to qualify the expressions of KRT13, KRT4 and 15-lipoxygenase-1 (15-LOX-1) in cultured ESCC cell lines with recombinant IL-13 treatment.ResultsIL-13 was expressed in the esophageal epithelium cells and ESCC tumor cells. High IL-13 expression in ESCC tumor cells predicted a good prognosis for patients. Recombinant human IL-13 raised KRT13 and 15-LOX-1 mRNA levels, but lowered KRT4 mRNA level 15-LOX-1 in ESCC cells in vitro.ConclusionsIn summary, our study suggests that IL-13 might improve the prognosis of ESCC by promoting the terminal differentiation of ESCC cells. This may offer potential new therapeutic target for early treatment of ESCC.     

Increased glucocorticoid receptor concentration in macrophage differentiation of myeloid leukemia cells with 12-O-tetradecanoylphorbol-13-acetate

HL60 cells, human promyelocytic leukemia cells, can be induced to differentiate into more mature myeloid forms by dimethyl sulfoxide (DMSO) or retinoic acid (RA) and into macrophage-like cells by 12-O-tetradecanoylphorbol-13-acetate (TPA) or related compounds. Macrophage differentiation of HL60 cells by TPA treatment induced a 3- to 4-fold increase in glucocorticoid receptor concentration per cell and a 2- to 3-fold increase in glucocorticoid receptor concentration per mg of protein. The ability of TPA derivatives to increase glucocorticoid receptor concentration paralleled their ability to induce macrophage differentiation. Macrophage differentiation of other myeloid leukemia cells by TPA treatment induced a 2- to 3-fold increase in glucocorticoid receptor concentration per cell. Exposure of T-lymphoblasts or erythroleukemia cells to TPA did not affect glucocorticoid receptor concentration. Myeloid differentiation of myeloid leukemia cells by DMSO or RA induced no significant change in glucocorticoid receptor concentration. The increase in glucocorticoid receptor concentration in macrophage differentiation of myeloid leukemia cells with TPA was considered to depend, not on TPA treatment, but on the process of macrophage differentiation. Further, glucocorticoid receptor concentration can be a sensitive marker of macrophage differentiation of myeloid leukemia cells.     

Recombinagenic activity of the phorbol ester 12-O-Metradecanoylphorbol-13-acetate in human lymphoblastoid cells

The potent mouse skin tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate(TPA) was examined for its mutagenic and recombinagenic activityat the heterozygous thymidine kinase (tk +/–) locus andthe hemizygous hypoxanthine phosphoribosyltransferase (hprt+/0) locus in the TK6 human lymphoblastoid cell line. TPA atconcentrations of 0.01–1.0 µg/ml induced a low frequencyof tk mutants showing the slow growth phenotype in a dose-dependentmanner, but few normal growth tk mutants or hprt mutants. Concentrationsof 1.0–10 µg/ml TPA induced all three types of mutants.The molecular structure of tk mutants arising spontaneouslyor induced by 1.0 and 10 µg/ml TPA was investigated bySouthern hybridization with a human tk cDNA probe: 86% of allmutants arising after incubation with 10 µg/ml TPA lostthe entire active tk allele, resulting in loss of heterozygosity(LOH), while 71% of spontaneously arising mutants showed LOH.Densitometric analysis indicated that the majority of LOH mutantsinduced by TPA were homozygous at the tk locus (retained twocopies of the mutant allele), consistent with the occurrenceof inter-chromosomal homologous recombination. These resultssupport the hypothesis that tumor promoters such as TPA mayincrease the rate of chromosomal mitotic recombination and hencefacilitate the segregation of recessive mutations. TPA may thusinduce a type of genetic instability during the process of tumorpromotion that involves enhanced recombinagenic activity.     

Comparison of effects of bryostatins 1 and 2 and 12-O-tetradecanoylphorbol-13-acetate on protein kinase C activity in A549 human lung carcinoma cells

Activators of protein kinase C (PKC), such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and bryostatins 1 and 2, inhibit the growth of A549 cells. At high concentrations the bryostatins do not affect cell growth. Here the hypothesis has been tested that modulation of A549 cell growth is the consequence of agent-induced changes in location or extent of cellular PKC activity. PKC activity was measured after semi-purification with nondenaturing polyacrylamide gel electrophoresis in the cytosol and the particulate fraction of A549 cells. When cells were exposed to TPA or mezerein, PKC activity underwent rapid and concentration-dependent translocation from the cytosol to the membrane. TPA at 0.1 microM or mezerein at 1 microM caused almost complete translocation within 30 min. Incubation with bryostatins 1 or 2 also led to enzyme translocation, which was, however, much weaker than that observed with the tumor promoters. Neither 4 alpha-phorboldidecanoate nor the synthetic diacylglycerols 1,2-sn-dioctanoylglycerol or 1-oleoyl-2-acetyl-sn-glycerol mimicked TPA in this way. Exposure of cells to TPA or the bryostatins for longer than 30 min caused the gradual disappearance of total cellular PKC activity. PKC downregulation was concentration dependent and complete after 24 h. A549 cells which had acquired temporary resistance toward the growth-arresting potential of TPA were completely devoid of any measurable PKC activity. The bryostatins were potent inhibitors of the binding of [3H]phorbol-12,13-dibutyrate to its receptors in intact cells, and the inhibition was dependent on bryostatin concentration. The results support the contention that PKC is involved in the mediation of growth inhibition caused by TPA or the bryostatins. However, the relationship between growth arrest and PKC translocation or downregulation seems to be a complex one.     

Establishment of a human leukemia subline resistant to the growth-inhibitory effect of 12-O-tetradecanoylphorbol 13-acetate (TPA) and showing non-P-glycoprotein-mediated multi-drug resistance.

We have previously reported that K562/ADM, a typical P-glycoprotein-mediated multi-drug-resistant cell line, is cross-resistant to the growth-inhibitory effect of 12-O-tetradecanoylphorbol 13-acetate (TPA) and non-TPA type tumor promoters. To elucidate the mechanism of cross-resistance to tumor promoters in K562/ADM, we have established a K562 subline resistant to TPA-induced growth inhibition by exposing K562 cells to N-methyl-N'-nitro-N-nitrosoguanidine for 24 hr followed by continuous exposure to TPA. A K562 subline resistant to the TPA-induced growth inhibition, termed K562/TPA, was selected by a limiting dilution technique. K562/TPA was more than 500-fold resistant to TPA compared with parental K562 cells. K562/TPA showed cross-resistance to etoposide, teniposide, adriamycin (ADM), vincristine, vindesine and 3-[(4-amino-2-methyl-5-pyrimidinyl)] methyl-1-(2-chloroethyl)-1-nitrosourea, but showed collateral sensitivity to cisplatin. Although K562/ADM was not cross-resistant to 3'-deamino-3'-morpholino-13-deoxo-10-hydroxycarminomycin (MX2), an anthracycline derivative, K562/TPA was cross-resistant to MX2. By Northern blot analysis, K562/TPA did not express MDR-1. Accumulation of ADM by K562/TPA was no lower than that of K562 although that of K562/ADM was 5-fold lower than K562. We examined the subcellular distribution of ADM by fluorescence microscopy. The fluorescence of ADM was located in the nucleus of K562 and mainly in the cytoplasm of K562/TPA and K562/ADM. The distribution of ADM in K562/TPA, however, was different from that in K562/ADM. These results suggested that K562/TPA had a non-P-glycoprotein-mediated multi-drug-resistance phenotype and that the mechanism of drug-resistance in this cell line might be explained by an alteration in the intracellular drug distribution.     

Ecto-5'-nucleotidase. II. Effect of 12-O-tetradecanoylphorbol 13-acetate on the expression of enzyme in normal and neoplastic B-cell lines

Immature B-cells, including B-cell lymphoma lines, are often deficient in ecto-5'-nucleotidase (5'-NT) activity. 12-O-Tetradecanoylphorbol 13-acetate (TPA) was shown to be capable of inducing maturation toward plasmacytoid-like cells in immunoglobulin (Ig)-secreting B-cell lines. An attempt was made to induce the enzyme in 5'-NT-negative B-cell lymphoma lines with TPA to clarify the relationship between 5'-NT and B-cell differentiation. After 3 days in the presence or absence of TPA, these cell lines were examined morphologically, and their 5'-NT activity, Ig secretion, surface Ig, and Ia, B1, and B2 antigens were estimated. Neither Ig secretion nor 5'-NT activity was induced by TPA in any of 4 nonsecreting cell lines studied. Ig secretion was significantly increased in 4 of 5 lg-secreting cell lines. Two of these inducible cell lines, JD 38 and ST 486, became positive for 5'-NT activity and acquired morphologic characteristics of plasma cells after culture with TPA. The lymphoma cell line JD 38 was transplanted into nude mice and gave rise to a solid tumor. Although the tumor cells remained negative for 5'-NT, they could be induced by TPA to express both the enzyme activity and plasmacytoid-like appearance. These data suggested that in the Ig-secreting B-cell lymphoma lines, there was an association between the inducibility of 5'-NT and the capacity of these cell lines to undergo plasma-cytoid-like transformation in response to TPA.