Generation of procoagulant activity by hairy cells in response to endotoxin and phorbol esters

Several investigators have suggested that hairy cells are neoplastic B lymphocytes. These cells, however, also share some biological properties with mononuclear phagocytes. A property of these cells is the capacity to generate procoagulant activity (PCA) in response to a variety of stimuli. In this study we investigated the PCA of peripheral blood hairy cells in 19 consecutive patients with hairy cell leukaemia (HCL). Monocyte-depleted blood mononuclear cells, tested immediately after isolation, expressed little, if any, activity. However, after exposure to endotoxin, a marked increase in PCA was observed (42.1 ± 8.7 vs 1.3 ± 0.2 units/5 × 10⁴ hairy cells). A significant correlation was found between the number of lymphocytes/hairy cell and the level of endotoxin-induced PCA suggesting that lymphocytes potentiate the procoagulant response of hairy cells. When stimulated with 12-0-tetradecanoyl-phorbol-13-acetate (TPA), patients' cells produced about 2–8 times more PCA than endotoxin-stimulated cells. The PCA induced by endotoxin and TPA was identified as tissue factor. These findings suggest some further relationship between hairy cells and monocytes.     

Induction of suppressor activity by tumor-promoting phorbol esters in primary cultures of lymph node cells

We have observed the induction of suppressor activity in primarycultures of lymphocytes by the tumor-promoting phorbol ester12-O-tetradecanoylphorbol-13-acetate (TPA). Suppressor activitywas detected as the ability of TPA-treated lympocytes to inhibitproliferation in mixed lymphocyte cul tures (MLC). Inductionof this activity was dependent on the dose of TPA and was maximalalter 12 h of incubation. Neither the induction of the activity,nor the activity itself was inhibited by indomethacin or interleukin2 (IL2). A comparison of addition of TPA directly to the MLC,addition of TPA pretreated cells as participants in the MLCand addition of cells treated with TPA to induce suppressoractivity suggested that the suppressor activity was only oneof the ways that TPA could inhibit proliferation. This suppressoractivi ty may account for some of the reported effects of TPAin immunological systems in vitro and suggests that suppressorcells could play a role in TPA-mediated promotion in vivo.     

Induction of a novel nuclear protein (p54) by phorbol esters in mouse erythroleukemia (Friend) cells

Tumor-promoting phorbol esters including 12-O-tetradecanoylphorbol-13-acetate (TPA), specific inhibitors for erythroid differentiation in mouse Friend cells, induce a newly identified nuclear protein with a molecular weight of 54,000 (p54) in Friend cells. Phorbol, an analogue of TPA with neither tumor-promoting nor differentiation-inhibitory activity, did not induce p54. In a variant cell line of Friend cells which exhibits resistance to TPA in erythroid differentiation, p54 was not induced by TPA. The induction of p54 by TPA was counteracted by erythroid-inducing agents including dimethyl sulfoxide, hexamethylenebisacetamide, and actinomycin D. Throughout these experiments, we have observed an inverse relationship between p54 induction and cellular potential for erythroid differentiation.     

Heterogeneity in TPA-induced differentiation of B-chronic lymphocytic leukemia cells: development of hairy cell or plasmacytoid features are time and dose dependent

Cells from 11 chronic lymphocytic leukemic (CLL) patients were induced to differentiate with various doses of tetradecanoyl phorbol-13-acetate (TPA) and the degree of induction was followed up to six days by measuring the expression of two surface membrane markers (SmIg and GP-70), Ig secretion, tartrate-resistant acid phosphatase (TRAP), and ultrastructural changes. The results indicate dose and time dependency of the TPA effect and a great heterogeneity in the response to TPA among cells from different CLL patients. Furthermore, the two main TPA-induced features, the "plasmacytoid" or "hairy cell" features depended on the dose and duration of treatment with the phorbolester. The plasmacytoid features were more frequently encountered at low doses (1 ng/ml) of TPA and were evident after short exposures to TPA (1-2 days). The hairy cell features were more obvious after incubation with higher doses of TPA (10-100 ng/ml) or at Day 6 with lower doses of TPA. The differentiation features measured, including cell morphology, surface membrane markers, Ig secretion, and TRAP staining, appeared to be independent of each other suggesting an autonomous pathway of differentiation for some of these features.     

Lineage relationship of chronic lymphocytic leukemia and hairy cell leukemia: studies with TPA

The tumor promoting agent TPA (phorbol ester; 1.6 X 10(-8)M) was used to induce the differentiation in vitro of B-chronic lymphocytic leukemia (B-CLL) cells from 14 untreated patients. The uninduced phenotype was SIg+, Mrbc+, RFT-1+, RFA-4-, FMC7-. After 72 h incubation with TPA, B-CLL cells became RFA-4+, FMC7+ and lost the capability of Mrbc rosetting. Large proportions of the "induced" cells also showed morphological and ultrastructural changes, such as undulating membranes and bleblike protusions and became strongly positive for tartrate resistant acid phosphatase (TRAP+) and also contained cytoplasmic immunoglobulins. These features are very similar to the features of hairy cell leukemia (HCL). These observations confirm previous clinical findings that B-CLL and HCL are related disorders of the B lineage. The development of "hairy" features in induced B-CLL and in HCL seems to be a malignancy-associated feature because the Mrbc+ normal B cells (B-CLL-equivalent cells) isolated from tonsil also develop TRAP positivity but no membrane aberrations.     

Phorbol esters and hairy cell leukemia: effects on cell morphology and surface membrane features and comparison with other B cell leukemias

Hairy cell leukemia (HCL) mononuclear cells were incubated with the phorbol ester TPA in an attempt to induce further maturation and were compared with B cell chronic lymphocytic leukemia, prolymphocytic leukemia, and non-Hodgkin's lymphoma cells. Morphology, surface features, membrane markers, tartrate-resistant acid phosphatase, and Ig secretion were examined. HCL cells spread and adhered firmly after TPA, producing elongated filopodia. Cells still retained ribosomal lamellar complexes, and increased numbers of dense bodies were seen. TPA enhanced the adherent and phagocytic properties of HCL cells, producing a modest increase in the expression of membrane Ig, GP-70, and Leu-M5 markers, tartrate-resistant acid phosphatase, and Ig secretion. Other neoplastic B cells behaved differently, forming readily detachable clumps without elongated filopodia. Maturation to plasma cells and hairy cell features were readily evident in all cases. These differences in growth patterns were consistent and may be used to distinguish HCL from other B cell neoplasias.     

Cytogenetic effects caused by phorbol ester tumor promoters in primary mouse keratinocyte cultures: correlation with the convertogenic activity of TPA in multistage skin carcinogenesis

The effects of the convertogenic (‘first-stage’)tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), thenon-convertogenic (‘second stage’) tumor promoter12-O-retinoylphorbol-13-acetate (RPA) and the non-promotingphorbol esters 4-O-MeTPA and 4--PDD on the chromosomes of mousekeratinocytes in primary cultures were investigated. In thesetarget cells of tumor promotion TPA caused severe numericaland structural chromosomal aberrations, which were evident aftertwo cell cycles and accumulated after multiple applications.Numerical aberrations were visible as hypo- and hyperdiploidy,with non-random loss or gain of specific chromosomes. The clastogeniceffects were evident as simple alterations such as gaps andbreaks, but more severe alterations such as tri- and quadriradialchromatid interchanges and ring chromosomes, as well as translocationscould be observed. The structural aberrations were nonrandomlydistributed in the genome and chromosomes no. 1, 2, 5, 6 and18 were preferentially involved in rearrangements. In additionto the aneuploidogenic, clastogenic and recombinogenic effectsinduced by TPA, short treatment with this tumor promoter wasefficient in producing cytogenetic equivalents of gene amplification,i.e. double minute chromosomes. The cytogenetic effects werenot merely due to cytotoxicity since they occurred after a lowTPA dose (10^–8 M) and did not considerably increase witha higher dose (10^–6 M). Moreover, at both dose levelscell cycle traverse of mouse keratinocytes was not drasticallyaltered. In contrast, the non-convertogenic tumor promoter RPAand the non-promoting phorbol esters 4--PDD and 4-O-MeTPA (atthe same dose level) did not cause any substantial chromosomalalterations. This discrepancy between the action of TPA andRPA suggests that effects which result in chromosomal alterationsin the target cells may be critical for the conversion stageof skin tumor promotion. This conclusion is supported by experimentswith substances such as antipain and eicosa-5,8,ll,14-tetraynoicacid which inhibit both tumor induction in initiated skin andthe cytogenetic alterations induced by TPA in cultured keratinocytes.These studies provide for the first time the possibility ofdifferentiating between convertogenic and non-convertogenictumor promoters in an in vitro assay using the target cellsof mouse skin carcinogenesis.     

Induction of an unusual type of shared phosphorylation in human and avian cells by tumor-promoting phorbol esters or transformation

Alterations in patterns of protein phosphorylation after exposure to phorbol esters were compared in chicken embryo fibroblasts and KD cells (a human fibroblast line) by two-dimensional gel electrophoresis. A substantial increase in phosphorylation was observed of a major, markedly acidic protein of pI = 4.5 in two-dimensional gels of each cell type. However, the apparent molecular weights of these phosphoproteins differed substantially in the two species with a molecular weight of 67,000 in chicken fibroblasts and one of 80,000 in human KD cells. Both phosphoproteins, termed 67K and 80K respectively, contained phosphoserine and a small amount of phosphothreonine, but no detectable level of phosphotyrosine. Tryptic and chymotryptic phosphopeptide maps of 67K were nearly identical to those of 80K. These results indicate that they are related molecules, even though considerably different in apparent molecular weight, and that the induction of phosphorylation of these closely related, major, acidic phosphoproteins by phorbol esters is conserved from avian to human cells. In chicken embryo fibroblasts infected by a temperature-sensitive mutant of Rous sarcoma virus, phosphorylation of 67K was found to be elevated at 36 degrees C (transformed phenotype) compared to 41.5 degrees C (normal). Although the function of these closely related 67K and 80K phosphoproteins is unknown, the elevated level of phosphorylation could be involved in some aspects of transformation, and the increase is mimicked by treatment with phorbol esters.     

Differentiation of human myeloid leukemic cells by phorbol esters: Correlation with tumor promotion

The effect of the plant diterpenes, phorbol derivatives and mezerein, on differentiation of various human myeloid leukemic cells to macrophages was determined. The results indicate that, within the group of phorbol esters tested, a correlation exists between the potency of the compounds as inducers of differentiation and their reported potency as tumor promoters. However, mezerein and 12-O-retinoylphorbol 13-acetate, which promote tumors only weakly or not at all, were found to be efficient inducers. The efficiency of all the active phorbol derivatives, including the weak inducers, also known to be weak promoters, could be potentiated by pretreatment of the cells with retinoids, compounds which have been reported to inhibit tumor promotion. Similar results were obtained in 3 different established cell lines, as well as in short-term cultures of cells obtained from patients with acute myeloid leukemia. The results suggest that the activities of the diterpenes as tumor promoters and inducers of differentiation are not necessarily linked. Moreover, certain conditions which are unfavorable for tumor promotion may not affect or even potentiate induction of differentiation.     

Induction of differentiation in chronic myelocytic leukaemia cells by the phorbolester TPA

The mononuclear peripheral blood cells from eight patients with chronic myelocytic leukaemia (CML) were incubated in cell suspension culture in the presence of the phorbolester 12-O-tetradecanoylphorbol 13-acetate (TPA). TPA caused the treated cells to adhere and to acquire morphological and functional features characteristic of macrophage-like cells. Using isoelectric focusing distinct changes in the isoenzyme profiles of carboxylic esterase, acid phosphatase, hexosaminidase and lactate dehydrogenase were detected in the TPA-exposed cells. Besides an increase in the number and staining intensity of isoenzymes of all enzymes, TPA triggered the new expression of a monocyte-specific esterase isoenzyme isoenzyme and of the tartrate-resistant acid phosphatase isoenzyme. The latter two isoenzymes represent further parameters of the monocyte/macrophage complex. The results indicate that immature leukaemic cells arrested along the granulocytic cell axis retain the ability to transform to macrophages.     

Rapid morphological and substratum adherence response by hairy cell and other human leukemic cells to phorbol ester tumor promoters

Human leukemic cells, maintained in tissue culture, responded differentially following exposure to the phorbol ester tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA). Short-term (0-2 h) TPA treatment of hairy cell leukemic cells (of presumed B-lymphocytic origin) resulted in the attachment of 5-100% of cells (depending on % leukemic mononuclear cells) to the culture dish and extension of long processes. Similar changes were also observed with cells from acute myelogenous, acute prolymphocytic and some acute monomyelocytic leukemic patients. Related non-promoters such as phorbol and 4-alpha-phorbol-12, 13-didecanoate, had no effect. In contrast, both chronic and acute lymphocytic, chronic myelogenous and acute promyelocytic leukemic cells showed no response to TPA over this short time period. Neither the adherence nor morphological changes were blocked by inhibitors of protein synthesis (cycloheximide) or glycosylation (tunicamycin), but the process formation was sensitive to inhibitors of microtubule formation such as colchicine. The effect was not reversible by TPA removal, and exposure to TPA for only 5 min resulted in a complete adherence and morphological induction within 60 min.     

Stimulation of cell proliferation and inhibition of differentiation expression by tumor-promoting phorbol esters in dog thyroid cells in primary culture

12-O-Tetradecanoylphorbol-13-acetate (TPA) and 4 beta-phorbol 12, 13-dibutyrate (PDBU) are potent tumor promoters and share several biological activities of epidermal growth factor (EGF). We have shown previously that EGF stimulates DNA synthesis and proliferation and inhibits TSH-induced markers of differentiation in dog thyroid follicle-derived primary cultures. Using this system, we have examined the biological action of TPA and PDBU in reference to that of EGF. Low concentrations (1.6-16 nM) and to a lesser extent higher concentrations (greater than 1.6 microM) of TPA and PDBU stimulated cell proliferation in a 1% serum, hormone-supplemented medium and triggered the DNA synthesis revealed by autoradiography in cells which were quiescent before stimulation in serum-free conditions. EGF, TSH, and dibutyryl cyclic adenosine 3':5'-monophosphate separately also induce DNA synthesis, but they produce little if any effects additive to those of TPA. In fact, TPA appeared to inhibit the mitogenic effects of EGF. Moreover like EGF, phorbol esters strongly inhibited in 2 days the morphological effects of TSH and basal and TSH-stimulated iodide transport capacity and thyroglobulin messenger RNA accumulation, two markers of thyroid differentiation. TPA also inhibited the expression of differentiation stimulated by dibutyryl cyclic adenosine 3':5'-monophosphate indicating a post-cyclic adenosine 3':5'-monophosphate site of action. TPA and EGF shared long-term morphological effects such as the induction of an elongated fusiform shape, but not acute effects. The thyroid cells progressively and spontaneously escaped both the mitogenic and differentiation-inhibiting effects of TPA and PDBU, while, as shown previously, these parameters are stably modified by continuous culture with EGF. This suggests specific desensitization processes to phorbol esters. As evidence is accumulating that phorbol esters act at least partly by stimulating the calcium-activated, phospholipid-dependent protein kinase C, our results shed light on the possible key role of this kinase in carcinogenesis and in the normal control of proliferation and expression of differentiation in the thyroid gland. Additionally they suggest that complex interactions occur between the mechanisms of action of EGF and of phorbol esters in the thyroid cell.     

Tumor induction in initiated mouse skin by phorbol esters and methyl methanesulfonate: correlation between chromosomal damage and conversion ('stage I of tumor promotion') in vivo

Using the multistage (initiation-conversion-promotion) protocol we have studied the effects of methyl methanesulfonate (MMS), a well-known alkylating and clastogenic agent, on tumor development in NMRI mouse skin in vivo. When topically applied in a dose up to 400 mumol MMS did not exhibit any initiating efficacy while under identical conditions chromosomal damage (mainly breaks and gaps) was induced in epidermis. A dose of 100 mumol MMS was found to be almost as clastogenic as 10 nmol of the convertogenic tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), whereas the non-convertogenic promoter 12-O-retinoylphorbol-13-acetate (RPA, 10 nmol) did not induce chromosomal aberrations in vivo. In combination with initiation by 7,12-dimethylbenz[a]anthracene (DMBA) and promotion by RPA, MMS (2 x 100 mumol) turned out to be a rather powerful convertogenic agent ('stage I tumor promoter'). This tumor-inducing efficacy of MMS was synergistically increased by simultaneous application of RPA. These results support the concept that the induction of chromosomal aberrations plays an important role in skin tumor development, i.e. in the conversion stage.     

Phospholipase activity in skin after application of phorbol esters and 3-methylcholanthrene

Topical administration of the promoter, 12-O-tetra-decanoylphorbol-13-acetate(TPA), is accompanied by an increased incorporation of [³H]arachidonicacid into mouse skin and a very significant activation of epidermal cell membrane phospholipase A₂ without affecting intracellularacid phospholipase. Similar enhancement in epidermal cell phospholipaseA₂ was observed after application of phorbol-12,13-didecanoateor 3-methyl-cholanthrene to mouse skin. A small but significantincrease in phospholipase A₂ activity is also seen after applicationof the nonpromoter irritant, acetic add. The elevated levelsof the prostaglandlns observed in mouse skin after topical applicationof promoters are probably triggered by the activation of thismembrane enzyme.     

Induction of protein kinase C down-regulation by the phorbol ester TPA in a calpain/protein kinase C complex.

Using a calpain/protein kinase C (PKC) complex, we were able to reproduce, in vitro, the induction of PKC down-regulation by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) which had been previously observed in cells. We show that TPA initiates this phenomenon by promoting a calpain-dependent conversion of PKC to the Ca2+ phospholipid-independent protein kinase M (PKM), at physiological calcium concentrations. This effect of TPA was dependent upon the presence of phosphatidylserine and was observed only when PKC was the substrate for the protease, inactivation of calpain by autolysis not being modified by the presence of TPA. Moreover, PKM generated from the calpain-PKC complex was resistant to calpain, even after addition of TPA. These results suggest that TPA induces a conformational change in PKC, increasing the affinity of the kinase for calpain and consequently permitting its proteolysis for the basal level of calcium in cells.     

Cytochemical analysis of acid hydrolases expression during phorbol diester (TPA)-driven differentiation of B-chronic lymphocytic leukaemia cells in vitro

Four acid hydrolases, acid phosphatase (AP), alpha-naphthyl acetate acid esterase (ANAE), beta-glucuronidase (BG) and N-acetyl-beta-glucosaminidase (NABG) were determined cytochemically in B-chronic lymphocytic leukaemia (B-CLL) cells exposed in vitro to the tumor promoter 12-0-tetradecanoyl phorbol 13 acetate (TPA). TPA, which has been previously shown to induce B-CLL cells to mature towards plasmacytoid cells, results in the progressive expression of the enzymes tested in the cytoplasm of malignant cells, in particular AP and ANAE. Furthermore, the sensitivity to inhibitors and the pattern of reactivity of ANAE provide evidence for an enzyme subtype normally restricted to plasma cells. Thus, acid hydrolases--some of which showing plasma cell type of activity--are expressed during B-CLL cells differentiation induced in vitro. These results confirm the value of cytochemistry in subtyping B-cell malignancies.