Phenylbutyrate induces cell differentiation and modulates Epstein-Barr virus gene expression in Burkitt's lymphoma cells.

Although Burkitt's lymphoma (BL) is a readily treated malignancy, recurrences, as well as disease arising in immunosuppressed patients, are notoriously resistant to conventional therapeutic approaches. The EBV is associated with a significant proportion of these lymphomas that evade immune surveillance through decreased expression of both viral and cellular antigens. Increasing the immunogenicity of BL cells may, therefore, represent a potentially beneficial therapeutic maneuver. Using in vitro models of EBV-transformed lymphoblastoid as well as BL cell lines, we demonstrate increased expression of genes coding for HLA class I and EBV latent proteins by the differentiation inducer phenylbutyrate (PB). The aromatic fatty acid also caused cytostasis associated with sustained declines in c-myc expression, a direct antitumor effect that was independent of the EBV status. We conclude, therefore, that differentiation therapy of BL with PB may lead to growth arrest with increased tumor immunogenicity in vivo. The findings may have clinical relevance because the in vitro activity has been observed with PB concentrations that are well tolerated and nonimmunosuppressive in humans, a desirable feature for the different patient populations afflicted with this disease.

Regulation of murine Max (Myn) parallels the regulation of c-Myc in differentiating murine erythroleukemia cells.

Max is a basic region-helix-loop-helix-leucine zipper protein that consists of two major isoforms, p22 (long form, Max-L) and p21 (short form, Max-S). These proteins are encoded by two [the 1.9- and the predominant 2.3-kilobase (kb) forms] of the five alternatively spliced max mRNA species. We now demonstrate that N,N'-hexamethylene bisacetamide-mediated differentiation of murine erythroleukemia cells leads to a pattern of biphasic down-regulation of the 1.9- and the 2.3-kb myn (murine max) mRNAs that closely parallels that which occurs for myc mRNA. In contrast, the p22/Myn-L and p21/Myn-S protein isoforms down-regulate in monophasic fashion. Unlike the short-lived myc mRNA, the myn message is quite stable. However, its half-life of 3-6 h is still consistent with the biphasic down-regulation that accompanies differentiation. Furthermore, unlike myc, the overexpression of which prevents differentiation, elevated max levels merely delay differentiation. Coincident with this is a delay in the second decline of c-myc mRNA. In N,N'-hexamethylene bisacetamide-induced cells blocked from differentiating by overexpression of c-, N- or L-myc, myn mRNA expression is constitutive. These findings suggest that myn may also be involved in differentiation.

Transfected wild-type and mutant max regulate cell growth and differentiation of murine erythroleukemia cells.

Max protein forms specific DNA-binding dimeric complexes with itself and with proteins of the c-myc gene family. A large volume of data has accumulated on the role of the c-myc proto-oncogene in cell proliferation, differentiation and tumorigenesis. To elucidate the role of max in regulating c-myc functions and the effect of both proteins on cell proliferation and differentiation, we transfected murine erythroleukemia (MEL) cells with a full-length wild-type (wt) human max gene and a mutant containing a double point mutation in the basic region (bm), which abolishes specific DNA binding. All clones expressing wt-max grow slowly, and the process of inducer-mediated differentiation is delayed. Furthermore, cells transfected with the mutated max exhibit growth retardation, accumulation in the G0/G1 phase of the cell cycle and spontaneous differentiation. Our findings are consistent with a model in which a large excess of wt-Max in the cells enhances the formation of Max-Max growth-suppressor complexes, while elevated bm-Max deprives the cell of growth-promoting Myc-Max heterodimers in a dominant-negative manner, presumably by inactivating endogenous Myc and Max.

Inhibition of murine erythroleukemia cell differentiation by normal and partially deleted c-myc genes.

In our study of normal and partially deleted myc genes we found that N-myc, similarly to L-myc, can substitute for c-myc and inhibit MEL cell differentiation. All of the known putative functional domains of c-myc seem to be required for this inhibition. It is conceivable that c-myc inhibits differentiation by a mechanism that is related to its normal role in the cell, possibly by regulating transcription of genes involved in growth promotion. As was previously found for all of the other known activities of c-Myc, the HLH and LZ dimerization motifs are absolutely necessary for inhibition of MEL cell differentiation. Heterodimerization of Myc with Max or Max-like proteins could be a prerequisite for such inhibition. It is, therefore, of interest to study the regulation of max in MEL cells expressing normal and deregulated myc genes.

Subcellular localization of heparanase in human neutrophils.

The subcellular localization of a heparan sulfate degrading endoglycosidase, heparanase, was studied in human neutrophils. Unstimulated cells were disrupted by nitrogen cavitation and fractionated on a Percoll density gradient into three components, separating the plasma membranes, specific granules, and azurophilic granules. Heparanase activity was measured by gel filtration analysis of 35S-labeled degradation fragments released from subendothelial extracellular matrix (ECM) or produced during incubation with soluble, ECM-derived, heparan sulfate proteoglycans. Heparanase activity was found mainly in fractions containing the specific granules; this activity was inhibited by heparin. Freezing and thawing was not needed for recovery of the enzyme from the subcellular fraction, confirming previous data about its ready release. The mechanism of the ready release of heparanase from the specific granules requires further investigation.

Regions within the c-Myc protein that are necessary for transformation are also required for inhibition of differentiation of murine erythroleukemia cells.

The c-, L-, and N-Myc nuclear phosphoproteins share several highly conserved regions that partially overlap putative functional domains of the c-Myc protein. All three myc oncogenes can cooperate with an activated ras gene to transform primary rat embryo cells (REC), and deregulated expression of c- and L-myc can block differentiation of murine erythroleukemia (MEL) cells. In the present study, we demonstrate that N-myc also can block MEL cell differentiation, and we identify regions within the c-Myc protein that are necessary for inhibition of MEL differentiation. C19 MEL cells were transfected with six human c-myc genes which were partially deleted in different areas of the coding region. Four of the genes lack sequences that overlap either the putative transcriptional activation domain, the helix-loop-helix motif, or the leucine zipper motif and were previously shown to have lost REC cotransforming activity (J. Stone, T. DeLange, G. Ramsay, E. Jakobovitz, J.M. Bishop, H. Varmus, and W. Lee, Mol. Cell. Biol., 7: 1697-1709, 1987). In this study, we demonstrate that they also fail to inhibit N,N'-hexamethylene-bis-acetamide-induced differentiation of MEL cells. In contrast, two partially deleted c-myc genes, one lacking a short NH2-terminal region and the other lacking 118 amino acids at the center of the coding region, which were fully active in REC cotransformation, also exhibited full activity associated with the former and only partial activity with the latter in blocking MEL differentiation. We conclude that the mutated genes tested in this study behave similarly in inhibition of MEL cell differentiation and in REC cotransformation.

Involvement of heparanase in tumor metastasis and angiogenesis.

The capacity of various blood-borne cells, whether normal or malignant, to extravasate was found to correlate with heparanase-mediated degradation of HS in subendothelial ECM. This degradation was stimulated by proteases or plasminogen and inhibited by native heparin and by various modified nonanticoagulant species of heparin. These heparins also induced a marked reduction in tumor cell metastasis and autoimmune diseases in experimental animals. Heparanase-mediated degradation of HS in ECM also released EC growth factors that are stored in ECM, most likely by high affinity binding to HS. Such growth factors were extracted from subendothelial ECM synthesized in vitro and from basement membranes of the cornea in vivo, and are structurally and functionally related to bFGF;bFGF binds to ECM and is readily released by incubation with either HS, heparin or low MW heparin fragments as well as by various normal and malignant cells and by heparanase-mediated degradation of ECM HS. In contrast, there was little or no release of growth-promoting activity upon incubation of ECM with hyaluronic acid, chondroitin sulfate or chondroitinase ABC. A model is proposed suggesting that regulation of capillary growth and neovascular response may result from displacement of an angiogenic protein (bFGF) from its storage sites within basement membranes.

Role of heparanase in platelet and tumor cell interactions with the subendothelial extracellular matrix.

Dissemination of neoplastic cells within the body involves invasion of blood vessels by tumor cells. Since platelets have been shown to contribute to this process, we studied the interaction in vitro of platelets and malignant cells with the vascular endothelium and its underlying basement membrane-like ECM. A metastatic subline (ESb) of the methylcholanthrene-induced DBA/2 T-lymphoma invaded the vascular endothelium at a higher rate than its parental nonmetastatic (Eb) subline. ESb cells also exhibited a much higher ability to degrade the proteoglycan scaffold of the ECM by means of a specific HS degrading endoglycosidase (heparanase). The interaction of platelets with this ECM was associated with platelet activation, aggregation, and degradation of HS by means of the platelet heparanase. Degradation of ECM-HS was facilitated by proteolytic activity that produced a more accessible substrate for further cleavage by heparanase. A similar enhancement was exerted by plasminogen via the activity of the tumor cells or ECM associated PAs. Heparin and chemically modified heparins that lack anticoagulant activity inhibited degradation of the ECM-HS by heparanase. Interaction of platelets and lymphoma cells with ECM covered with vascular endothelial cells was investigated by SEM and by determination of ECM-HS degradation products. SEM studies demonstrated that platelets may adhere to minor gaps between adjacent endothelial cells and degrade the ECM-HS. Platelets were also shown to recruit lymphoma cells into these interendothelial gaps, suggesting that by binding to ECM and release of heparanase, platelets may play an active role in tumor cell invasion and metastasis. Our observation that nonanticoagulant heparins may interfere with heparanase-mediated degradation of ECM-HS suggests a potential therapeutic use for such heparins in neoplastic disorders.

Inhibition of heparanase-mediated degradation of extracellular matrix heparan sulfate by non-anticoagulant heparin species.

Incubation of human platelets, human neutrophils, or highly metastatic mouse lymphoma cells with sulfate-labeled extracellular matrix (ECM) results in heparanase-mediated release of labeled heparan sulfate cleavage fragments (0.5 less than Kav less than 0.85 on Sepharose 6B). This degradation was inhibited by native heparin both when brought about by intact cells or their released heparanase activity. Degradation of heparan sulfate in ECM may facilitate invasion of normal and malignant cells through basement membranes. The present study tested the heparanase inhibitory effect of nonanticoagulant species of heparin that might be of potential use in preventing heparanase mediated extravasation of bloodborne cells. For this purpose, we prepared various species of low-sulfated or low-mol-wt heparins, all of which exhibited less than 7% of the anticoagulant activity of native heparin. N-sulfate groups of heparin are necessary for its heparanase inhibitory activity but can be substituted by an acetyl group provided that the O-sulfate groups are retained. O-sulfate groups could be removed provided that the N positions were resulfated. Total desulfation of heparin abolished its heparanase inhibitory activity. Heparan sulfate was a 25-fold less potent heparanase inhibitor than native heparin. Efficiency of low-mol-wt heparins to inhibit degradation of heparan sulfate in ECM decreased with their main molecular size, and a synthetic pentasaccharide, representing the binding site to antithrombin III, was devoid of inhibitory activity. Similar results were obtained with heparanase activities released from platelets, neutrophils, and lymphoma cells. We propose that heparanase inhibiting nonanticoagulant heparins may interfere with dissemination of bloodborne tumor cells and development of experimental autoimmune diseases.

Involvement of both heparanase and plasminogen activator in lymphoma cell-mediated degradation of heparan sulfate in the subendothelial extracellular matrix.

The effect of plasminogen on the ability of highly metastatic ESb mouse lymphoma cells to degrade heparan sulfate (HS) in the subendothelial extracellular matrix (ECM) was studied. A metabolically sulfate-labeled ECM was incubated with the lymphoma cells, and labeled degradation products were analyzed by gel filtration on Sepharose 6B. Heparanase-mediated release of low-Mr (0.5 less than Kav less than 0.85) HS cleavage products was stimulated fourfold in the presence of plasminogen. Incubation of plasminogen alone with the ECM resulted in its conversion into plasmin, which released high-Mr (Kav less than 0.33) labeled proteoglycans from the ECM. Heating the ECM (80 degrees C, 1 hr) abolished its ability to convert plasminogen into plasmin, yet plasminogen stimulated, through its activation by the ESb plasminogen activator, heparanase-mediated release of low-Mr HS fragments. Heparin inhibited both the basal and plasminogen-stimulated degradation of HS side chains but not the total amount of labeled material released from the ECM. In contrast, aprotinin inhibited the plasminogen-stimulated release of high- as well as low-Mr material. In the absence of plasminogen, degradation of heated ECM by ESb cells was completely inhibited by aprotinin, but there was only a partial inhibition of the degradation of native ECM and no effect on the degradation of soluble HS proteoglycan. These results demonstrate that proteolytic activity and heparanase participate synergistically in the sequential degradation of ECM HS and that the ESb proteolytic activity is crucial for this degradation when the ECM-associated protease is inactivated. Plasminogen may serve as a source for the proteolytic activity that produces a more accessible substrate to the heparanase.

Degradation of heparan sulfate in the subendothelial extracellular matrix by a readily released heparanase from human neutrophils. Possible role in invasion through basement membranes.

Freshly isolated human neutrophils were investigated for their ability to degrade heparan sulfate proteoglycans in the subendothelial extracellular matrix (ECM) produced by cultured corneal and vascular endothelial cells. The ECM was metabolically labeled with Na2(35S)O4 and labeled degradation products were analyzed by gel filtration over Sepharose 6B. More than 90% of the released radioactivity consisted of heparan sulfate fragments 5-6 times smaller than intact heparan sulfate side chains released from the ECM by either papain or alkaline borohydride. These fragments were sensitive to deamination with nitrous acid and were not produced in the presence of either heparin or serine protease inhibitors. In contrast, degradation of soluble high molecular weight heparan sulfate proteoglycan, which was first released from the ECM, was inhibited by heparin but there was no effect of protease inhibitors. These results indicate that interaction of human neutrophils with the subendothelial ECM is associated with degradation of its heparan sulfate by means of a specific, newly identified, heparanase activity and that this degradation is facilitated to a large extent by serine proteases. The neutrophil heparanase was readily and preferentially released (15-25% of the cellular content in 60 min) by simply incubating the cells at 4 degrees C in the absence of added stimuli. Under these conditions, less than 5% of the cellular content of lactate dehydrogenase, lysozyme, and globin degrading proteases was released. Further purification of the neutrophil heparanase was achieved by its binding to heparin-Sepharose and elution at 1 M NaCl. It is suggested that heparanase activity is involved in the early events of extravasation and diapedesis of neutrophils in response to a threshold signal from an extravascular inflamed organ.

Sequential degradation of heparan sulfate in the subendothelial extracellular matrix by highly metastatic lymphoma cells.

A highly metastatic variant (ESb) of a methylcholanthrene-induced T lymphoma elaborates a heparan sulfate (HS) degrading endoglycosidase (heparanase) to a much higher extent than its non-metastatic parental subline (Eb). Whereas a serum-free medium conditioned by either subline contained a trypsin-like serine protease, heparanase activity was detected only in the ESb-conditioned medium (CM). ESb CM was incubated with a naturally produced, sulfate-labelled subendothelial extracellular matrix (ECM) or with a soluble, high-MW labelled proteoglycan first released from the ECM by incubation with Eb CM or with the partially purified ESb protease. Sulfate labelled degradation products were analyzed by gel filtration on Sephrose 6B. The optimal pH for degradation of ECM-bound HS was 6.2 as compared to pH 5.2 for degradation of the soluble proteoglycan. Heparanase-mediated degradation of both ECM-bound and soluble HS was inhibited by heparin. Addition of either trypsin, plasmin or to a lower extent, the purified ESb protease, stimulated between 5- and 20-fold the ESb CM-mediated degradation of ECM-bound HS but had no effect on heparanase-mediated degradation of the soluble proteoglycan. This stimulation was inhibited in the presence of heparin or protease inhibitors. These results indicate that both a protease and heparanase are involved in the ESb-mediated degradation of ECM-bound HS and that one enzyme produces a more accessible substrate for the next enzyme. This sequential cleavage is characteristic of degradation of a multimolecular structure such as the subendothelial ECM and hence cannot be detected in studies with its isolated constituents.

Lymphoma cell-mediated degradation of sulfated proteoglycans in the subendothelial extracellular matrix: relationship to tumor cell metastasis.

Cloned lines of the methylcholanthrene-induced DBA/2 low-metastatic T-lymphoma Eb line and its highly metastatic variant ESb line were compared for the ability to degrade proteoglycans in the subendothelial extracellular matrix (ECM) produced by cultured endothelial cells. The ECM was metabolically labeled with Na2(35)SO4, and the tumor cell-mediated release of labeled degradation products was analyzed by gel filtration. More than 90% of the labeled material released upon incubation of ESb cells with the ECM, either when exposed or covered with vascular endothelial cells, was in the form of low-Mr, heparan sulfate-containing fragments (Mr approximately 10(4)) compared to high-Mr sulfated proteoglycans (mostly excluded from Sepharose 6B) released by incubation with the low-metastatic Eb cells. The same high- and low-Mr degradation products were obtained by incubation of the ECM with a serum-free medium conditioned by the low (Eb)- and high (ESb)-metastatic sublines, respectively. The high-Mr proteoglycans released by incubation of the ECM with Eb-conditioned medium was further degraded into Mr 10(4) glycosaminoglycan fragments upon a subsequent incubation with ESb-conditioned medium. These fragments were smaller than glycosaminoglycan side chains released by treatment of the ECM with papain or alkaline borohydride, suggesting an ESb-specific endoglycosidase activity. The higher ability of the ESb over the Eb cells to solubilize the glycosaminoglycan scaffolding of the sub-endothelial ECM may, among other properties, facilitate their hematogenous dissemination and extravasation.