Hyaluronidase enhancement of TNF-mediated cell death is reversed by TGF-beta 1

Nan-Shan Chang

Laboratory of Molecular Immunology, Guthrie Research Institute, Guthrie Medical Center, Sayre, Pennsylvania 18840

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Both hyaluronidase and transforming growth factor (TGF)-beta 1 play a significant role in the development of prostate cancer. In this study, the regulation of tumor necrosis factor (TNF)-mediated cell death by hyaluronidase and TGF-beta 1 was investigated. Preexposure of L929 fibroblasts, prostate LNCaP cells, and epithelial Mv 1 Lu cells to hyaluronidase for a minimum of 12 h resulted in significant enhancement of cell death by TNF. Phosphorylation of p42 and p44 mitogen-activated protein (MAP) kinases was found by stimulation of L929 cells with hyaluronidase for 30 min, indicating that the Raf/MAP kinase-extracellular signal-regulating protein kinase (MEK)/MAP kinase pathway was activated. However, blocking the activation of upstream MAP kinase kinase (MEK 1 and 2 kinase) by PD-98059 failed to inhibit the hyaluronidase-enhanced TNF killing of cells, suggesting that hyaluronidase-mediated degradation of extracellular matrix and membrane components may elicit multiple signaling pathways. As a potent stimulator of extracellular matrix protein synthesis, TGF-beta 1 blocked the hyaluronidase-enhanced death of L929 and LNCaP cells mediated by TNF. TGF-beta 1 activated protein-tyrosine kinases in L929 cells, in which the tyrosine kinase inhibitors lavendustin A and tyrphostin blocked the activation as well as the TGF-beta 1 inhibition of hyaluronidase effects. Functional antagonism was also observed between hyaluronidase and TGF-beta 1 in cell growth regulation. For example, TGF-beta 1-mediated suppression of epithelial Mv 1 Lu cell growth was abolished by hyaluronidase. Overall, it is demonstrated in this study that hyaluronidase reciprocally antagonized TGF-beta 1 in the modulation of cell proliferation and TNF-mediated death.

transforming growth factor-beta 1; tumor necrosis factor; apoptosis; L929 cells; LNCaP cells

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

AS A MATRIX-DEGRADING ENZYME, hyaluronidase participates in cancer progression, invasion, and angiogenesis. Various cancer cells, such as Wilms' tumor, produce hyaluronidase (22, 29). Elevation of hyaluronidase levels is associated with prostate cancer progression (18). Expression of hyaluronidase by tumor cells induces angiogenesis in vivo (17). Notably, the growth of murine lung carcinoma and melanoma is influenced by Hyal-1, a locus determining hyaluronidase levels and polymorphism (11).

As opposed to the function of hyaluronidase, transforming growth factor (TGF)-beta is a potent stimulator of extracellular matrix protein synthesis (25). Moreover, TGF-beta increases tumorigenicity in vivo by suppressing the local immune system (13, 15). Overexpression of TGF-beta is frequently associated with the development of benign prostatic hyperplasia to malignant prostate cancer (27, 28) and breast cancer progression (1, 33).

In this study, TGF-beta 1 was found to reciprocally counteract hyaluronidase function in the regulation of cell proliferation and tumor necrosis factor (TNF)-mediated cell death. Both in vitro and in vivo studies have shown that destruction of extracellular matrix by hyaluronidase increases cancer cell susceptibility to chemotherapeutic drugs (2, 10, 16, 24, 26). As determined in this study, pretreatment of L929 fibrosarcoma (6, 7), prostate LNCaP (31) and lung epithelial Mv 1 Lu (23) cells with hyaluronidase for at least 12 h, followed by exposure to TNF, resulted in increased cell death.

TGF-beta 1 protects L929 cells from TNF killing (6). Interestingly, TGF-beta 1 inhibited the hyaluronidase-mediated enhancement of TNF killing. Conversely, hyaluronidase counteracted TGF-beta 1-mediated growth inhibition of epithelial Mv 1 Lu cells (23). How TGF-beta 1 counteracted hyaluronidase induction of TNF sensitivity in L929 cells was investigated.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell lines. TNF-sensitive L929 cells were cultured in 10% newborn calf serum-RPMI 1640 medium as previously described (6, 7). Mink lung epithelial Mv 1 Lu cells (23) and human prostate cancer LNCaP cells (31) were cultured according to the instructions of the American Type Culture Collection (Rockville, MD).

TNF cytotoxicity assays. TNF cytotoxicity assays were performed as previously described (5-7). Briefly, 100-µl aliquots of L929 cells (2.5 × 105 cells/ml) were dispensed onto 96-well microtiter plates (Corning, Corning, NY), cultured for 24 h, treated with bovine testicular hyaluronidase (0-200 U/ml; Sigma) for 12-24 h, and exposed to recombinant TNF-alpha (500-4,000 U/ml; Genzyme, Boston, MA) for 16-24 h. To determine the extent of death, the cells were fixed and stained with crystal violet (2% in 50% methanol) and analyzed in an automatic microtiter plate reader for optical density (OD) at 560 nm (SLT Labinstruments, Research Triangle Park, NC). Compared with each hyaluronidase-treated or untreated cell group, the extent of cell death in the corresponding TNF-treated cell group (hyaluronidase-treated or untreated cells) was calculated as follows: %Cell death = [(OD from control cells - OD from TNF-treated cells) / OD from control cells] × 100. The extent of hyaluronidase-mediated enhancement of TNF cytotoxicity was also calculated: %Enhancement = [(%TNF killing of hyaluronidase-stimulated cells / %TNF killing of control cells) - 1] × 100 (5-7). Unless otherwise indicated, data are presented as means ± SD (n = 8). As indicated, the extent of cell death was also confirmed by using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS tetrazolium) proliferation assay kit (Promega, Madison, WI). Conversion of the MTS tetrazolium into a soluble formazan by viable cells was measured as 490-nm OD (4). Similar experiments were performed using Mv 1 Lu and LNCaP cells.

The extent of TNF-mediated apoptotic cell death was also confirmed by determination of internucleosomal DNA fragmentation, as measured in agarose gel electrophoresis described previously (5).

Chemicals used to modulate the hyaluronidase-mediated enhancement of TNF killing were the mitogen-activating protein (MAP) kinase-extracellular signal-regulating protein kinase (MEK) 1 and 2 kinase inhibitor PD-98059 (21), the tyrosine kinase inhibitors tyrphostin (12) and lavendustin A (20), and the protein kinase C (PKC) inhibitor H-7 (14). These chemicals were utilized in cotreatment of L929 cells with hyaluronidase for indicated durations, and the cells were then exposed to TNF.

Functional antagonism of TGF-beta 1. TGF-beta 1 regulation of hyaluronidase-mediated enhancement of L929 cell death by TNF was examined. L929 cells were pretreated with hyaluronidase in the presence or absence of purified human platelet TGF-beta 1 (0.25-2 ng/ml; Collaborative Research, Bedford, MA) for 16-24 h, followed by removal of the culture supernatants and exposure to TNF for 16-24 h. Similarly, the hyaluronidase-pretreated L929 cells (16-24 h) were exposed to TGF-beta 1 for 1-7 h, followed by removal of culture supernatants and incubation with TNF for 16-24 h, or the hyaluronidase-pretreated cells were exposed simultaneously to TGF-beta 1 and/or TNF for 16-24 h. Also, TGF-beta 1 (0.25-2 ng/ml; in 50 µl of RPMI 1640 medium containing 2% albumin) was precoated onto 96-well microtiter plates for 9 h in a humidified 37°C incubator (nondry coating with lids on), after which the plates were washed twice with phosphate-buffered saline (PBS) and L929 cells were seeded. These cells were cultured overnight, treated with hyaluronidase for 24 h, and subsequently exposed to TNF for 16-24 h.

Additional cytokines used in the testings were recombinant TGF-beta 2 (Oncogene Science, Uniondale, NY), TGF-beta 3 (Oncogene Science), interferon-gamma (Genzyme), interleukin (IL)-4 (PeproTech, Rocky Hill, NJ), IL-6 (a reference standard from the National Cancer Institute, Bethesda, MD), IL-10 (PeproTech), and IL-13 (PeproTech).

Western blotting. To examine whether hyaluronidase activated the Raf/MEK/MAP kinase pathway, L929 cells in petri dishes were treated with hyaluronidase (100 U/ml) for 0, 0.5, 1, 2, or 4 h, followed by washing with cold PBS and lysis of the cells with a lysis buffer (6). Western blotting was performed as described (8). Briefly, 10 µg of the cell lysates were subjected to reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroblotting of the separated proteins onto a Zeta-probe nylon membrane (Bio-Rad Laboratories, Hercules, CA), and probing with specific polyclonal immunoglobulin G antibodies against a synthetic phosphopeptide of p42 and p44 MAP kinases (New England BioLab, Beverly, MA).

Hyaluronidase blocking of TGF-beta 1-mediated growth inhibition. TGF-beta 1 is known to restrict the proliferation of Mv 1 Lu cells (23). To examine the functional antagonism between TGF-beta 1 and hyaluronidase, Mv 1 Lu cells were treated with TGF-beta 1 in the presence or absence of hyaluronidase for 24 and 48 h. The extent of cell proliferation was examined by both crystal violet staining and the MTS assay.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Hyaluronidase enhancement of TNF- and serum deprivation-mediated apoptosis. Pretreatment of L929 cells with hyaluronidase for 16 h at 37°C, followed by removal of hyaluronidase and exposure to TNF (1,000 U/ml) for 24 h, resulted in increased cell death or apoptosis (Fig. 1). These results were observed by staining the cells with crystal violet, and comparable results were obtained using the MTS proliferation assay (data not shown). The enhanced cell death also occurred when the hyaluronidase-pretreated L929 cells were exposed to TNF with the continuous presence of hyaluronidase. Hyaluronidase increased L929 cell growth by ~5-10% in 16 h (Fig. 1). Time course studies demonstrated that development of the enhanced TNF killing required pretreatment of L929 cells with hyaluronidase for a minimum of 12 h (data not shown). TNF-mediated apoptotic cell death was verified by analysis of internucleosomal DNA fragmentation. Compared with control cells, the hyaluronidase-pretreated cells, when challenged with TNF for 6 h, had increased DNA fragmentation (Fig. 1). A higher concentration of TNF (4,000 U/ml) was used in this short-term assay.


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Fig. 1.   Hyaluronidase enhancement of tumor necrosis factor (TNF)-mediated internucleosomal DNA fragmentation and death of L929 cells. Left: L929 cells were pretreated with hyaluronidase for 16 h, followed by removal of hyaluronidase and addition of TNF-alpha (1,000 U/ml) for 24 h. Extent of cell death was determined by crystal violet staining method and comparable results were obtained by MTS assays (data not shown). Compared with controls, percentages of hyaluronidase-mediated increase in cell death are shown in parentheses. Effect of hyaluronidase on L929 cell growth is also shown (without TNF treatment; open circle ). Right: under similar conditions, L929 cells were pretreated with hyaluronidase for 16 h and then treated with or without TNF-alpha (4,000 U/ml) for 6 h in absence of hyaluronidase. Nuclear DNA was isolated from cells and electrophoresed in a 2% agarose gel. +, TNF-treated cells; -, cells without TNF treatment. DNA molecular size standard is in lane at left.

When prostate LNCaP cells were pretreated with hyaluronidase for 24 h and subsequently exposed to TNF, TNF killing of the cells was increased by ~540-1,200% (or 5.4- to 12-fold; Fig. 2). However, without pretreatment, cotreatment of LNCaP cells with hyaluronidase and TNF failed to increase the TNF killing (data not shown). Normal lung epithelial Mv 1 Lu cells were also responsive to hyaluronidase induction of TNF sensitivity (during pretreatment) by approximately onefold increase.


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Fig. 2.   Hyaluronidase (HAase) enhancement of TNF killing of prostate LNCaP cells. LNCaP cells were pretreated with hyaluronidase for 24 h and then exposed to TNF-alpha for 24 h in absence of hyaluronidase. Enhanced cell death for LNCaP cells is ~540-1,200%.

Hyaluronidase also enhanced serum deprivation-mediated growth inhibition. Shown in the Fig. 3 is the increased growth inhibition caused by pretreatment of L929 cells with hyaluronidase for 24 h, followed by culturing under serum-free conditions for 24 h.


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Fig. 3.   Hyaluronidase enhancement of serum deprivation-mediated growth inhibition of L929 cells. L929 cells were pretreated with hyaluronidase for 24 h, followed by removal of hyaluronidase and culturing under serum-free conditions for 24 h.

Hyaluronidase activation of the Raf/MEK/MAP kinase pathway in L929 cells. Hyaluronidase-mediated activation of p42 and p44 MAP kinases was examined. Exposure of L929 cells to hyaluronidase for 30 min resulted in activation of p42 and p44 MAP kinases of the Raf/MEK/MAP kinase pathway, as observed in the Western blotting using the specific antibodies against a synthetic tyrosine-phosphopeptide of p42 and p44 MAP kinases (Fig. 4A). Nonetheless, blocking the activation of MEK 1 and 2 kinases (upstream activators of p42 and p44 MAP kinases) by the specific inhibitor PD-98059 failed to abolish the hyaluronidase enhancement of TNF killing. Indeed, PD-98059 alone inhibited TNF killing of L929 cells, whereas hyaluronidase reversed this inhibitory effect and restored the sensitivity of L929 cells to TNF (Fig. 4B). During the 48-h culture period, PD-98059 significantly inhibited L929 cell growth, whereas hyaluronidase reversed the growth inhibitory effect of PD-98059 (Fig. 4C).


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Fig. 4.   Activation of p42 and p44 mitogen-activated protein (MAP) kinases in hyaluronidase-stimulated L929 cells and failure of specific MAP kinase-extracellular signal-regulating protein kinase inhibitor PD-98059 in blocking hyaluronidase-enhanced TNF killing. A: L929 cells were treated with hyaluronidase (100 U/ml) for 0, 0.5, 1, and 2 h, followed by lysis of cells. Cell lysates (10 µg) were subjected to reducing SDS-polyacrylamide gel electrophoresis and Western blotting using immunoglobulin G antibodies against a phosphopeptide of p42 and p44 MAP kinases (see MATERIALS AND METHODS). B: L929 cells were pretreated with hyaluronidase (50 U/ml) and PD-98059 for 24 h, followed by coexposure to TNF-alpha (4,000 U/ml) for an additional 24 h. PD-98059 alone blocked TNF killing of L929 cells, which was reversed by hyaluronidase. C: during 48-h treatment, PD-98059 suppressed L929 cell growth and growth inhibition was reversed by hyaluronidase.

TGF-beta 1 inhibition of hyaluronidase enhancement of TNF killing. TGF-beta 1 was found to inhibit hyaluronidase-mediated enhancement of TNF killing. When L929 cells were pretreated with hyaluronidase for 22 h, followed by exposure to TGF-beta 1 for 1.5 h and subsequent treatment with TNF for 16-24 h, the hyaluronidase-enhanced TNF killing was blocked by TGF-beta 1 (Fig. 5A). Similar results were obtained by exposure of the hyaluronidase-pretreated L929 cells to TGF-beta 1 for 2-7 h (data not shown). Exposure of the hyaluronidase-pretreated L929 cells to both TNF and TGF-beta 1 for 16-24 h also resulted in abrogation of the effects of hyaluronidase. Growth of L929 cells overnight in 96-well plates that had been precoated with TGF-beta 1, followed by treatment with hyaluronidase for 24 h, also resulted in blocking of the hyaluronidase enhancement of TNF killing by TGF-beta 1 (Fig. 5B). TGF-beta 1 also inhibited TNF-mediated nuclear DNA fragmentation in both control and hyaluronidase-pretreated cells (Fig. 5C).


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Fig. 5.   Transforming growth factor (TGF)-beta 1 suppression of TNF-mediated internucleosomal DNA fragmentation and death of both control and hyaluronidase-treated L929 cells. A: L929 cells were pretreated with hyaluronidase for 22 h, then exposed to TGF-beta 1 (2 ng/ml) for 1.5 h, and subsequently treated with TNF-alpha (1,000 U/ml) for 16-24 h. B: 96-well plates were precoated with TGF-beta 1 (0-2 ng/ml) at 37°C for 9 h (nondry coating with lid on), followed by 2 washes with phosphate-buffered saline and seeding of L929 cells overnight. These cells were treated with hyaluronidase (100 U/ml) for 24 h, washed once with medium, and exposed to TNF-alpha (1,000 U/ml) for 24 h. C: under similar conditions, L929 cells were pretreated with hyaluronidase for 22 h, then exposed to TGF-beta 1 (2 ng/ml) for 1.5 h, and subsequently treated with TNF-alpha (4,000 U/ml) for 6 h. Nuclear DNA was isolated and electrophoresed in a 2% agarose gel. DNA molecular size standard is in lane at left.

Blocking of the hyaluronidase effects by TGF-beta 1 was also observed in LNCaP cells (Fig. 6). LNCaP cells were pretreated with hyaluronidase for 24 h, followed by coincubation with TGF-beta 1 for 2 h, then removal of hyaluronidase and TGF-beta 1, and exposure to TNF for 24 h.


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Fig. 6.   TGF-beta 1 suppression of hyaluronidase enhancement of TNF killing of LNCaP cells. LNCaP cells were pretreated with hyaluronidase (100 U/ml) for 24 h, then coincubated with TGF-beta 1 for 2 h, and finally treated with TNF-alpha (4,000 U/ml) for 24 h (in absence of both hyaluronidase and TGF-beta 1).

Under similar experimental conditions, cytokines, including interferon-gamma (0.1-100 ng/ml), IL-4 (1-1,000 pg/ml), IL-6 (10-10,000 U/ml), IL-10 (0.1-100 ng/ml), and IL-13 (1-1,000 pg/ml), were not capable of blocking the hyaluronidase effects (data not shown). Both TGF-beta 2 and TGF-beta 3 were ~50% less effective than TGF-beta 1 in blocking the hyaluronidase effects.

Blocking of TGF-beta 1 effects by protein-tyrosine kinase inhibitors. Previously, I have shown that protein-tyrosine kinase inhibitors, such as lavendustin A and tyrphostin, inhibit TGF-beta 1 function in protecting L929 cells against TNF-mediated cell death and that TGF-beta 1 stimulates protein-tyrosine phosphorylation in L929 cells (6). In this study, both lavendustin A and tyrphostin blocked the counteractive effect of TGF-beta 1 against hyaluronidase. When L929 cells were pretreated with hyaluronidase for 16-24 h and subsequently exposed to TGF-beta 1 in the presence of lavendustin A or tyrphostin for 7 h, the TGF-beta 1 inhibition of the hyaluronidase-increased TNF killing was abolished (Fig. 7). In contrast, H-7, a PKC inhibitor, was less effective in blocking the TGF-beta 1 effect (Fig. 7). Both lavendustin A and tyrphostin could not abolish the effect of hyaluronidase in increasing TNF sensitivity in L929 cells (data not shown).


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Fig. 7.   Blocking of TGF-beta 1 effects by tyrosine kinase inhibitors. L929 cells were pretreated with hyaluronidase (100 U/ml) for 24 h, then treated with TGF-beta 1 (2 ng/ml) and/or kinase inhibitors (10 µM), including lavendustin A (LA), tyrphostin (tyrpho), and H-7, for 7 h, and finally exposed to TNF alone (1,000 U/ml) for 24 h. Lavendustin and tyrphostin are tyrosine kinase inhibitors, and H-7 is an inhibitor of protein kinase C.

Hyaluronidase reversal of TGF-beta 1-mediated suppression of Mv 1 Lu cell growth. Finally, the functional antagonism between hyaluronidase and TGF-beta 1 was observed in the regulation of cell growth. In agreement with other observations (23), treatment of Mv 1 Lu cells with TGF-beta 1 resulted in inhibition of cell proliferation (Fig. 8A). This growth inhibitory effect of TGF-beta 1 was reversed by hyaluronidase (Fig. 8A). TGF-beta 1 inhibited Mv 1 Lu cell growth but failed to induce apoptosis in these cells, as determined by nuclear DNA fragmentation analysis (Fig. 8B).


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Fig. 8.   Hyaluronidase reversal of TGF-beta 1-mediated suppression of Mv 1 Lu cell growth. A: Mv 1 Lu cells, cultured in Eagle's minimum essential medium + 10% fetal bovine serum, were treated with TGF-beta 1 in presence or absence of hyaluronidase (100 U/ml) for 24 h. Relative cell numbers were determined by staining cells with crystal violet. Similar results were obtained using MTS assays. B: no internucleosomal DNA fragmentation was observed when Mv 1 Lu cells were treated with TGF-beta 1 and/or hyaluronidase (100 U/ml) for 24 h. Lane 1, control cells; lane 2, cells treated with hyaluronidase; lane 3, cells treated with TGF-beta 1 (4 ng/ml); lane 4, cells treated with TGF-beta 1 (2 ng/ml); lane 5, cells treated with TGF-beta 1 (4 ng/ml) and hyaluronidase; lane 6, cells treated with TGF-beta 1 (2 ng/ml) and hyaluronidase. DNA molecular size standard is in lane at left.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

In this study, functional antagonism between hyaluronidase and TGF-beta 1 was observed in the modulation of cell proliferation and TNF-mediated cell death. As an extracellular matrix degrading enzyme, hyaluronidase increased TNF-mediated killing of L929 fibroblasts, prostate LNCaP cells, and epithelial Mv 1 Lu cells. In contrast, as a potent stimulator of extracellular matrix protein synthesis, TGF-beta 1 blocked the hyaluronidase-mediated enhancement of TNF killing in these cells. Conceivably, one of the mechanisms by which TGF-beta 1 blocked the hyaluronidase effects is TGF-beta 1 restoration of the damaged extracellular matrix by hyaluronidase.

The physical integrity of extracellular matrix is important in protecting cancer cells from attack by anticancer drugs and the immune system. Thus treatment of cancer cells with hyaluronidase, either in vivo or in vitro, increases their susceptibility to anticancer drugs (2, 10, 16, 24, 26). However, these observations are in contrast to my previous report that, when L929 cells were pretreated with hyaluronidase for 12-24 h, the cells resisted killing by TNF and anti-Fas antibodies in the presence of anticancer drugs such as actinomycin D, doxorubicin, daunorubicin, and cycloheximide (7). In this study, however, pretreatment of L929 and LNCaP cells with hyaluronidase for 12-24 h resulted in increased TNF sensitivity without the presence of these anticancer drugs. These observations indicate that hyaluronidase-mediated de novo protein synthesis is essential for conferring TNF susceptibility in these cells. Indeed, by metabolic labeling we have demonstrated seven protein species induced by hyaluronidase in L929 cells (N.-S. Chang, N. Joki, J. Mattison, E. Chu, and M. Ou, unpublished observations). Conceivably, one of these proteins is capable of enhancing TNF killing in L929 cells.

Additionally, a likely scenario that accounts for the increased TNF sensitivity in L929 and LNCaP cells is due to the release of growth factors from the extracellular matrix on digestion with hyaluronidase. The extracellular matrix is an excellent reservoir for growth factors such as fibroblast growth factor. Presumably, these growth factors enhance L929 cell proliferation and increase their TNF susceptibility, since in most cases TNF targets proliferating cells. Thus the role of growth factors from the extracellular matrix in conferring TNF susceptibility in certain cancer cells remains to be established.

Although degradation of extracellular matrix hyaluronic acid and chondroitin sulfate occurred in hyaluronidase-treated cells, exogenous glycosaminoglycans, including hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate and heparin, failed to block the hyaluronidase effects (data not shown). Notably, as a stabilizer of extracellular matrix, the hyaluronic acid-binding protein inter-alpha -inhibitor (9) blocks hyaluronidase induction of TNF sensitivity in L929 cells (Chang et al., unpublished observations). This observation indicates that extracellular matrix proteins participate in conferring TNF resistance in L929 and probably other cells.

Previously, I have shown that hyaluronidase fails to alter the expression of TNF receptors and the receptor binding affinity for TNF in L929 cells (7). Furthermore, hyaluronidase-pretreated L929 cells, with subsequent exposure to TNF and/or actinomycin D, exhibit no significant changes in the expression of apoptosis regulatory proteins (7), such as Bcl-2 (19), Bcl-x (3), Ich-1 (32) and the RNA-binding protein TIAR (30). Thus these proteins are not involved in the hyaluronidase-mediated enhancement of TNF killing in L929 cells.

Not all the cancer cells can be induced by hyaluronidase to increase their TNF susceptibility. For example, TNF-resistant prostate DU 145, ovarian Me-180, and monkey kidney COS-7 cells were refractory to hyaluronidase induction of TNF sensitivity. The underlying mechanism is unknown. However, the differences in the responsiveness of prostate LNCaP and DU 145 cells to hyaluronidase induction of TNF sensitivity suggest that cellular genetic variations and mutations, culture conditions, and cell origins contribute to their differential responsiveness to TNF and/or hyaluronidase.

The Raf/MEK/MAP kinase pathway was activated when L929 cells were treated with hyaluronidase for 30 min, as evidenced by the phosphorylation of p42 and p44 MAP kinases. However, inhibition of the upstream MEK 1 and 2 kinase activation by PD-98059 failed to abolish the hyaluronidase effects. These observations suggest that multiple signaling pathways may be transduced into cells by digestion of the cells and surrounding extracellular matrix with hyaluronidase.

Most interestingly, PD-98059 inhibited L929 cell proliferation and rendered these cells TNF resistant. These inhibitory effects were reversed by hyaluronidase. Thus this observation further supports the notion that additional signaling pathways are elicited by hyaluronidase. In addition to PD-98059, I have examined a variety of protein tyrosine and serine/threonine kinase inhibitors in blocking the hyaluronidase effects. These inhibitors included lavendustin A, tyrphostin, herbimycin A, H-7, and G-490 (a selective inhibitor of JAK-2 kinase). Nonetheless, these inhibitors were not able to block the hyaluronidase effects. Thus hyaluronidase-elicited signaling transduction remains to be established.

Hyaluronidase partially enhanced serum growth factor-mediated proliferation of L929 cells, whereas on withdrawal of serum the hyaluronidase-pretreated L929 cells had an increased retardation of growth. The continuous presence of serum supports the cells to enter rounds of cell cycles, and hyaluronidase enhances the cell cycle progression, probably by inducing de novo protein synthesis. On withdrawal of serum growth factors, the hyaluronidase-pretreated L929 cells probably enter growth arrest in the G1 phase faster than untreated starved cells. These hyaluronidase-pretreated L929 cells will undergo apoptosis as long as the serum growth factors are continuously absent. Although it is not yet tested, it is reasonable to postulate that activation of caspase proteins, the cell death proteases, is enhanced in hyaluronidase-pretreated L929 cells, compared with control cells.

How hyaluronidase supports prostate cancer progression is unknown. On the basis of our data from L929 and other cells, hyaluronidase stimulates cancer cell growth, and these proliferating cells are the targets for anticancer drugs, such as actinomycin D, doxorubicin, and daunorubicin, and for TNF. However, development of drug resistance in cancer cells is not only dependent on the expression of the multiple drug resistance gene but is also related to hyaluronidase secretion. Although cotreatment of cancer cells with hyaluronidase and anticancer drugs increases the drug-mediated killing (2, 10, 16, 24, 26), my unpublished observations showed that, when L929 and LNCaP cells were pretreated with hyaluronidase for 24 h, these cells became resistant to killing by actinomycin D, doxorubicin, and daunorubicin. Thus hyaluronidase-secreting cancer cells may indeed resist killing by anticancer drugs, thus allowing their progression.

TGF-beta 1 restoration of the hyaluronidase-destroyed extracellular matrix may contribute in part to the inhibition of hyaluronidase effects. As a potent builder of extracellular matrix, TGF-beta 1 abrogated the hyaluronidase enhancement of TNF killing of L929 and LNCaP cells. Previously, I reported that TGF-beta 1 rapidly increases protein tyrosine phosphorylation in L929 cells, and that these cellular tyrosine phosphorylated proteins interrupt the TNF killing pathway (6). Accordingly, treatment of L929 cells with TGF-beta 1 in the presence of tyrosine kinase inhibitors such as lavendustin A and tyrphostin abolished the TGF-beta 1 antagonism of hyaluronidase.

Functional antagonism also demonstrated that hyaluronidase blocked TGF-beta 1-mediated growth inhibition of Mv 1 Lu cells. Similar results were also observed in COS-7 and Me-180 cells (data not shown). Although the underlying mechanism remains to be established, my observations raise an intriguing question regarding how overproduction of both hyaluronidase and TGF-beta contributes to the progression and metastasis of prostate cancer. Conceivably, a balanced production of hyaluronidase and TGF-beta 1 by prostate cancer is important for their development.

Overall, in this study, the in vitro functional antagonism between hyaluronidase and TGF-beta 1 is demonstrated. Whether hyaluronidase and TGF-beta 1 act synergistically or antagonistically in vivo remains to be established.

    ACKNOWLEDGEMENTS

I thank Dr. Lynda Bonewald (University of Texas Health Science Center, San Antonio, TX) for carefully reviewing the manuscript. The technical assistance of Nicole Joki and Jeffery Mattison is appreciated.

    FOOTNOTES

This research was supported by the Wendy Will Case Cancer Fund, National Cancer Institute Grants R01-CA61879 and R55-CA64423, and the Guthrie Foundation for Education and Research.

Received 27 June 1997; accepted in final form 25 August 1997.

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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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