Human Salivary Gland Adenocarcinoma Cell Research Articles

ヒト顎下腺由来腺癌細胞株(HSG) レチノイン酸レセプターと レチノイン酸レスポンスエレメント における との結合

ヒト顎下腺由来腺癌細胞株(HSG) レチノイン酸レセプターと レチノイン酸レスポンスエレメント における との結合

5′-Nitro-indirubinoxime induces G1 cell cycle arrest and apoptosis in salivary gland adenocarcinoma cells through the inhibition of Notch-1 signaling

Background 5′-Nitro-indirubinoxime (5′-NIO) is a new derivative of indirubin that exhibits anti-cancer activity in a variety of human cancer cells. However, its mechanism has not been fully clarified. Methods Human salivary gland adenocarcinoma (SGT) cells were used in this study. Western blot and RT-PCR analyses were performed to determine cellular Notch levels. The cell cycle stage and level of apoptosis were analyzed using flow cytometry analysis. Results 5′-NIO significantly inhibited the mRNA levels of Notch-1 and Notch-3 and their ligands (Delta1, 2, 3, and Jagged-2) in SGT cells. Immunocytochemistry analysis showed that 5′-NIO specifically decreased the level of Notch-1 in the nucleus. In addition, 5′-NIO induced G1 cell cycle arrest by reducing levels of CDK4 and CDK6 in SGT cells. Using flow cytometry and immunoblotting analysis, we found that 5′-NIO induces apoptosis following the secretion of cytochrome c and the activation of caspase-3 and caspase-7. Intracellular Notch-1 overexpression led to a decrease in G1 phase arrest and an inhibition of 5′-NIO-induced apoptosis. Conclusion These observations suggest that 5′-NIO induces cell cycle arrest and apoptosis by down-regulating Notch-1 signaling. General significance This study identifies a new mechanism of 5′-NIO-mediated anti-tumor properties. Thus, 5′-NIO could be used as a candidate for salivary gland adenocarcinoma therapeutics.

Refractory Factors in Head and Neck Cancer: ATP Binding Cassette Transporters Expressed in Head and Neck Cancer Cell Lines

The aim of the present study was to clarify whether ATP binding cassette transporters are refractory factors in head and neck cancer chemotherapy. For in vitro and in vivo chemotherapeutic studies, we employed a human salivary gland adenocarcinoma cell line (HSY) and a human oral squamous cell carcinoma cell line (SCCSK) with vincristine (VCR) at clinically equivalent doses. Western blot analysis, reverse transcription-polymerase chain reaction, in vivo evaluation in xenograft models inoculated with cultured carcinoma cell line and drug efflux analysis were performed. VCR-treated SCCSK and HSY cells, as well as xenografted SCCSK and HSY cells in tumor-bearing nude mice, were found to express MDR1/ABCB1 and MRP1/ABCC1. In addition to MDR1 and MRP1 mRNA, HSY/VCR and its cloned cells expressed MRP7/ABCC10 mRNA, but SCCSK/VCR did not express MRP7. Furthermore, drug resistance to VCR and docetaxel decreased in HSY/VCR in the presence of a competitive MRP7 inhibitor, 17-beta-estradiol-(17-beta-D-glucuronide). These results indicate that MDR1 and MRP1 expression are refractory factors in head and neck cancer chemotherapy and suggest that induction of MRP7 expression is involved in drug resistance in salivary gland adenocarcinomas.

Mechanism of Fas-mediated cell death and its enhancement by TNF-alpha in human salivary gland adenocarcinoma cell line HSG.

Fas-mediated cell death in a human salivary gland adenocarcinoma cell line (HSG) was induced by treatment of the cells with agonistic anti-Fas antibody (CH-11), and this cell death was enhanced by pretreatment with tumor necrosis factor alpha (TNF-alpha). The mode of cell death was apoptosis, because it was accompanied by caspase activation and the cleavage of poly(ADP-ribose) polymerase. The TNF-alpha treatment of the cells increased the expression of Fas, which was accompanied by the activation of nuclear factor kappaB (NFkappaB). These results suggest that the enhancement of the apoptosis caused by TNF-alpha resulted from increased sensitivity of the HSG cells to CH-11-mediated apoptosis due to induction of Fas protein by TNF-alpha via the activation of NFkappaB. In order to elucidate the apoptosis signaling pathway, we examined the effect of various caspase inhibitors on the apoptosis induced by CH-11. Fas-mediated apoptosis of HSG cells was slightly inhibited by the caspase-9 inhibitor although it was mainly inhibited by that for caspase-8. Based on this finding, we consider CH-11-induced apoptosis in HSG cells to be mainly mediated by the type I death signaling pathway that is caused by a caspase cascade initiated by the activation of caspase-8 at the death-inducing signaling complex (DISC).

Involvement of HSP90 in Anti-Fas-induced Apoptosis Signaling in the Human Salivary Gland Adenocarcinoma Cell Line HSG

It has been reported that heat shock protein (HSP) 90 is involved in the regulation of signaling cascades including those resulting in cell proliferation and apoptosis. However, little is known about how HSP90 affects apoptosis signaling.In this study, using a specific inhibitor of HSP90, geldanamycin (GDM), we investigated the relationship between HSP90 and anti-Fas antibody-induced apoptosis in HSG cells and concomitantly examined the apoptosis-inducing ability of GDM. We also sought to identify the target proteins of HSP90. When HSG cells were treated with GDM, a time-dependent increase in cell death was found. From the morphological features, the positive TdT-mediated dUTP nick end labeling (TUNEL) staining, and the cleavage of poly (ADP-ribose) polymerase (PARP), we concluded that the induced cell death was apoptotic. The pretreatment with GDM prior to that with anti-Fas antibody (CH-11) significantly increased the cell death as compared with that obtained with GDM or CH-11 alone. Further, prolonged incubation with GDM prominently enhanced the cell death. The induced cell death was also apoptotic. The transfection of HSG cells with recombinant HSP90α significantly inhibited the CH-11- and GDM-induced apoptosis. From the inhibition and overexpression experiments on HSP90 using GDM treatment and transfection with HSP90, respectively, we showed that HSP90 has an anti-apoptotic activity toward HSG cells. Immunoprecipitation with anti-human HSP90 antibody and subsequent Western blotting analysis of the precipitates detected bands for Caspase-8 and FADD-like ICE inhibitory protein (FLIP), which is known to regulate Fas cascade. Therefore, Caspase-8 and FLIP were shown to be target proteins of HSP90. These results suggest that HSP90 inhibits apoptosis by associating with Caspase-8 and FLIP and negatively regulating their functions.

Effect of salivary gland adenocarcinoma cell-derived α-N-acetylgalactosaminidase on the bioactivity of macrophage activating factor

The aim of this study was to clarify the effects of alpha-N-acetylgalactosaminidase (alpha-NaGalase) produced by human salivary gland adenocarcinoma (SGA) cells on the bioactivity of macrophage-activating factor (GcMAF). High exo-alpha-NaGalase activity was detected in the SGA cell line HSG. HSG alpha-NaGalase had both exo- and endo-enzyme activities, cleaving the Gal-GalNAc and GalNAc residues linked to Thr/Ser but not releasing the [NeuAc2-6]GalNac residue. Furthermore, GcMAF enzymatically prepared from the Gc protein enhanced the superoxide-generation capacity and phagocytic activity of monocytes/macrophages. However, GcMAF treated with purified alpha-NaGalase did not exhibit these effects. Thus, HSG possesses the capacity to produce larger quantities of alpha-NaGalase, which inactivates GcMAF produced from Gc protein, resulting in reduced phagocytic activity and superoxide-generation capacity of monocytes/macrophages. The present data strongly suggest that HSG alpha-NaGalase acts as an immunodeficiency factor in cancer patients.

ヒト顎下腺由来腺癌細胞株（ＨＳＧ）における抗Ｆａｓ抗体誘導アポトーシスシグナルへのＨＳＰ９０の関与

ヒト顎下腺由来腺癌細胞株（ＨＳＧ）における抗Ｆａｓ抗体誘導アポトーシスシグナルへのＨＳＰ９０の関与

Expression of cAMP Response Element Binding Protein (CREB)‐Binding Protein (CBP) and the Implication in Retinoic Acid‐Inducible Transcription Activation in Human Salivary Gland Adenocarcinoma Cell Line HSG

In the process of retinoic acid (RA) signaling, retinoic acid receptor interacts with a coactivator complex composed of various transcription cofactors such as CREB‐binding protein (CBP)/p300 and p160 family member proteins represented by steroid receptor coactivator‐1 (SRC‐1)/NCoA1 and p300/CBP cointegrator protein (p/CIP)/ACTR. In order to investigate the relationship of CBP to the RA signaling in a human salivary gland (HSG) adenocarcinoma cell line, we examined the expression of CBP in the cells. Immunoprecipitation and immunoblotting of the nuclear extract of HSG cells with anti‐human CBP antibody showed a specific 270‐kDa band, indicating the expression of CBP in HSG cells. The immunocytochemical analysis confirmed the nuclear localization of CBP. The transfection of HSG cells with a luciferase reporter plasmid harboring an RA‐response element at the 5′‐upstream region of the reporter gene increased RA‐dependent luciferase activity approximately 3‐fold. Co‐transfection with a CBP‐expression plasmid and the luciferase reporter gene enhanced the RA‐dependent transcription activation approximately 10‐fold. The immunoprecipitates obtained with anti‐CBP antibody exhibited a histone acetyl‐transferase (HAT) activity 2‐fold higher than that obtained with the control antibody, whereas the HAT activity of the immunoprecipitates with anti‐SRC‐1 and anti‐p/CIP, which were used as comparisons, were only a little increased. The RA treatment had no effect on the level of HAT activity except in the case of using the immunoprecipitate obtained with anti‐RARα, in which case it increased the activity. These findings indicate that CBP expressed in HSG cells mediates the RA‐inducible growth and differentiation‐regulating transcription activation in concert with the retinoic acid receptors.

Thermoradiotherapy combined with p53 gene therapy of human salivary gland adenocarcinoma cell line

We report on thermoradiotherapy combined with p53 gene therapy in the human salivary gland adenocarcinoma cell line HSG. HSG cells were successfully infected at a rate of 62.3% with MOIs of 20 of either AxCAip53 or AxCAiLacZ. The AxCAiLacZ did not inhibit the survival of the HSG cells. The survival fractions of AxCAip53-infected HSG cells were lower than those of the AxCAiLacZ-infected HSG cells, but there was no significant difference (p=0.30). AxCAip53 decreased the survival rates of thermotherapy (43 degrees C; p=0.084 and 44 degrees C; p=0.18), radiotherapy (6 Gy; p=0.20) and thermoradiotherapy (6 Gy plus 43 degrees C; p=0.24 and 6 Gy plus 44 degrees C; p=0.96), but there were no significant differences. In comparing the survival rates of thermoradiotherapy with those of thermotherapy and radiotherapy, thermoradiotherapy, regardless of the combination with p53 gene therapy, demonstrated actual survival rates lower than theoretical survival rates based on survival rates of thermotherapy multiplied by survival rates of radiotherapy. This result indicates that thermoradiotherapy is effective in the treatment of HSG cells. Thermoradiotherapy combined with p53 gene therapy was the most effective therapy among the combinations of therapies demonstrating that thermoradiotherapy combined with p53 gene therapy may be a useful tool in the treatment of HSG cells.

Thermochemotherapy of a human salivary adenocarcinoma cell line

We report on thermochemotherapy in a human salivary gland adenocarcinoma cell line. Hyperthermia reduced the survival rate to 50 and 20% by heating at 43 degrees C for 40 and 60 min, respectively, and is by itself useful in human salivary gland carcinoma treatment. Adriamycin, cisplatin, and mitomycin C can possible be used clinically, while bleomycin and 5-fluorouracil cannot, to treat this carcinoma. The optimal temperature was considered to be 41 degrees C in adriamycin, 42 degrees C in cisplatin, 37 degrees C in mitomycin C, and 42 degrees C in bleomycin in the thermochemotherapy. Thermochemotherapy is a useful tool in the treatment of human salivary gland carcinoma cells, but it is necessary to select the best anticancer drugs and the optimal temperature for optimal success using this treatment.

Inhibition of retinoic acid-inducible transcription by COUP-TFI in human salivary gland adenocarcinoma cell line HSG

Human salivary gland adenocarcinoma cells (HSG) express nuclear receptors, all-trans-retinoic acid (at-RA) receptors (RARs), and retinoid X/9-cis-retinoic acid (9-c-RA) receptors (RXRs). In order to investigate whether the endogenous RARs or RXRs of HSG cells can induce transcription activation, the thymidine kinase promoter (TK)-driven luciferase reporter gene containing the retinoic acid response element (RARE), of RARbeta, betaRARE2-TK-Luc, was transfected into HSG cells and ligand-dependent transcription activation was examined. Luciferase activity of cell lysate increased by the treatment with either at-RA or 9-c-RA. Co-transfection of RARalpha and (or) RXRalpha-expression plasmids with the reporter gene enhanced the luciferase activity, suggesting that endogenous RARs and RXRs work as ligand-dependent transfactors in HSG cells. Reverse transcriptase - polymerase chain reaction analysis revealed that HSG cells express chicken ovalbumin upstream promoter - transcription factor I (COUP-TFI). Co-transfection of COUP-TFI-expression plasmid suppressed the at-RA-induced transcription activation of the reporter gene. Similar results were shown using a chromatin-integrated reporter gene system, using a stably transfected beta-RARE2-TK-beta-galactosidase (beta-Gal) reporter gene. The at-RA-dependent increase in the beta-Gal expression was completely inhibited by COUP-TFI. The transfection of antisense oligonucleotide of COUP-TFI squelched the RA-dependent growth inhibition induced by RAR-RXR heterodimers. Conclusively, RARs and RXRs of HSG cells are functional and play roles as transactivators in at-RA-sensitive processes such as the proliferation or differentiation of cells. COUP-TFI very likely regulates these processes by repressing the functions of these transactivators.

Inhibition of retinoic acid-inducible transcription by COUP-TFI in human salivary gland adenocarcinoma cell line HSG

Inhibition of retinoic acid-inducible transcription by COUP-TFI in human salivary gland adenocarcinoma cell line HSG

Decrease in gamma-actin expression, disruption of actin microfilaments and alterations in cell adhesion systems associated with acquisition of metastatic capacity in human salivary gland adenocarcinoma cell clones.

In order to clarify how cytoskeletons and adhesion systems change through acquisition of metastatic capacity in a cancer cell, we examined the expressions of beta- and gamma-actin, the morphology of actin microfilaments and focal contacts, and also the expression of vinculin in a salivary gland adenocarcinoma cell clone cl-1, which acquired metastatic capacity, in comparison with its original clone HSGc lacking metastatic ability. Two-dimensional gel electrophoresis of Triton-insoluble fractions and combined Western blot analysis by immunostaining with anti actin-isoform antibodies showed that the expression of gamma-actin was somewhat lower than that of beta-actin in HSGc, and cl-1 expressed a comparable amount of beta-actin to HSGc, whereas gamma-actin expression by cl-1 was far less than that by HSGc. Northern blot analysis demonstrated that there was little difference in the level of beta-actin mRNA between HSGc and cl-1, while the level of gamma-actin was markedly decreased in cl-1 as compared with HSGc. In terms of morphology, cl-1 cells showed disruption of actin microfilaments and a decrease in the size and number of focal contacts on the cell surface. Furthermore, cl-1 showed decreased expression of vinculin, which became obscured even at the end of actin microfilaments. These results demonstrated that a decrease in gamma-actin, disruption of actin microfilaments, and suppression of focal contacts as well as vinculin take place in the transformation from a non-metastatic condition to a metastatic one in the human salivary gland adenocarcinoma cell clones. Thus, it was strongly suggested that these changes contribute to a decrease in cell adhesiveness and an increase in cell motility, which is probably a major cause for acquisition of metastatic potential.

Expression of retinoid X receptors and COUP-TFI in a human salivary gland adenocarcinoma cell line

The growth of the adenocarcinoma cell line derived from human salivary gland (HSG) is regulated by all-trans-retinoic acid (t-RA), which binds to its specific receptor, retinoic acid receptors (RARs), located in the nucleus, and thereby transactivates target genes. In this study, we examined the binding characteristics of the nuclear extract of HSG cells to the retinoic acid response element (RARE) compared with those of in vitro translated RAR alpha and retinoid X receptor alpha (RXR alpha), a heterodimeric partner of RAR alpha. Gel shift analysis using anti-RAR alpha and anti-RXR alpha antibodies revealed that the translated RAR alpha bound to RARE as a heterodimer with RXR alpha. In contrast, the binding of the nuclear extract of HSG cells to RARE showed a heterogeneous pattern, suggesting the existence of several species of RXRs as well as RARs in the nuclei of HSG cells. We therefore tried to clone these putative RXRs by the polymerase chain reaction using degenerated oligonucleotide primers conserved across the RXR family. The DNA sequencing of the recombinant clones revealed the expression of RXR alpha and RXR beta. In addition, chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI), which is also an RXR family member, was cloned. To evaluate the transcriptional activity of RARs and RXRs endogenously expressed in HSG cells, we performed a transient transfection analysis. When HSG cells were transfected with a luciferase reporter plasmid containing two repeats of either the RARE of the RAR beta gene or that of cellular retinol-binding protein II gene, positioned upstream of a thymidine kinase promoter fused to the luciferase sequence, a 2-3-fold induction of luciferase activity was observed in both cases. These results suggest that RARs and RXRs endogenously expressed in HSG cells were transcriptionally active in vivo. Thus, our findings showed that RXR alpha, RXR beta, and COUP-TFI are expressed in HSG cells and suggest that these molecules function as heterodimeric partners of RARs and (or) competitive repressors for RAREs and are involved in cellular responses mediated by retinoids.

Enhancement of bFGF export associated with malignant progression of human salivary gland cell clones.

Using 2 in vitro systems in which normal human salivary gland cells can be immortalized by transfection with SV40 DNA (NS-SV-DC), and in which a non-metastasizing human salivary gland adenocarcinoma cell clone (HSGc) can be converted to biologically aggressive and metastasizing cancer cells, we analyzed the relationship between the expression of basic fibroblast growth factor (bFGF) and malignant phenotype in human salivary gland cell clones. By enzyme immunoassay, intracellular contents of bFGF were equally detected in all the cell clones, while bFGF contained in serum-free conditioned medium of each cell clone was different: HSGc and metastasizing cell clones, respectively, released 2- and 10-fold more bFGF into media than did NS-SV-DC. Immunoblot analysis also revealed a similar result as that detected by enzyme immunoassay. By Northern blot analysis, bFGF mRNA expression was consistently detected in all the cell clones examined. We then quantitated bFGF contents in sera of tumor-bearing mice by enzyme immunoassay. Although sera from nude mice bearing an HSGc tumor showed significantly higher amounts of bFGF than those of control mice, bFGF concentrations in sera gradually decreased after removal of the tumor. For metastasizing cell clones, serum concentrations of bFGF were 2.2- to 2.6-fold higher than those of HSGc. In addition, bFGF amounts in sera after removal of the tumors were significantly higher in mice bearing metastasizing cell clone tumors compared with those of HSGc tumor-bearing mice. Our results, therefore, indicate that increase in bFGF release occurs along with the progression of human salivary gland cancer cells and that serum bFGF may be useful for evaluation of tumor presence.

Expression of retinoid X receptors and COUP-TFI in a human salivary gland adenocarcinoma cell line

The growth of the adenocarcinoma cell line derived from human salivary gland (HSG) is regulated by all-trans-retinoic acid (t-RA), which binds to its specific receptor, retinoic acid receptors (RARs), located in the nucleus, and thereby transactivates target genes. In this study, we examined the binding characteristics of the nuclear extract of HSG cells to the retinoic acid response element (RARE) compared with those of in vitro translated RAR alpha and retinoid X receptor alpha (RXR alpha ), a heterodimeric partner of RAR alpha . Gel shift analysis using anti-RAR alpha and anti-RXR alpha antibodies revealed that the translated RAR alpha bound to RARE as a heterodimer with RXR alpha . In contrast, the binding of the nuclear extract of HSG cells to RARE showed a heterogenous pattern, suggesting the existence of several species of RXRs as well as RARs in the nuclei of HSG cells. We therefore tried to clone these putative RXRs by the polymerase chain reaction using degenerated oligonucleotide primers conserved across the RXR family. The DNA sequencing of the recombinant clones revealed the expression of RXR alpha and RXR beta . In addition, chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI), which is also an RXR family member, was cloned. To evaluate the transcriptional activity of RARs and RXRs endogenously expressed in HSG cells, we performed a transient transfection analysis. When HSG cells were transfected with a luciferase reporter plasmid containing two repeats of either the RARE of the RAR beta gene or that of cellular retinol-binding protein II gene, positioned upstream of a thymidine kinase promoter fused to the luciferase sequence, a 2-3-fold induction of luciferase activity was observed in both cases. These results suggest that RARs and RXRs endogenously expressed in HSG cells were transcriptionally active in vivo. Thus, our findings showed that RXR alpha , RXR beta , and COUP-TFI are expressed in HSG cells and suggest that these molecules function as heterodimeric partners of RARs and (or) competitive repressors for RAREs and are involved in cellular responses mediated by retinoids. Key words: retinoid X receptor, retinoic acid receptor, retinoic acid response element, COUP-transcription factor (COUP-TF).

Tenascin: growth and adhesion modulation--extracellular matrix degrading function: an in vitro study.

Tenascin (TN), a recently characterised extracellular matrix protein, largely confined to the process with the development of embryo in areas of epithelial-mesenchymal interactions and in areas where there are morphogenetic movements and tissue patterning, has a highly restricted expression in adult tissues. The expression of TN is enhanced in a variety of human neoplastic lesions. However, function(s) and molecular mechanisms of enhanced expression in neoplastic lesions remain unclear. We employed human tongue carcinoma cells (SCCKN), human salivary gland adenocarcinoma cells (SGT-1), normal mouse embryonic fibroblasts (NIH3T3-3) and K-ras-2 transformed fibroblasts (Cle-H3) in an in vitro study to elucidate the biological roles of TN. In in vitro studies, all the cell lines examined had enhanced secretion of TN in the presence of transforming growth factor-beta in a dose-dependent manner and TN itself was found to possess a growth-enhancing activity. Moreover, studies on adhesion of the cell lines on coated substrates of fibronectin (FN), laminin (LN), tenascin (TN), TN/FN and TN/LN showed that all the cells adhere and spread well on FN and LN. However, on TN they attach poorly and remain rounded. The relative concentrations of TN and FN affected the cellular adhesion and morphology. In SCCKN and SGT-1, but not in NIH3T3 and Cle-He3 fibroblasts, a higher concentration of TN inhibited cellular adhesion on fibronectin, suggesting that cells attach poorly on TN, it may interfere with the action of fibronectin, and the relative concentrations of TN, FN or LN may affect cellular adhesion and morphology which may differ in different cell types. When TN was added in the growth medium of exponentially growing cells, the cells lost their cell to cell contact and were seen to be separating. The presence of these extracellular matrix proteins were further tested to determine whether they could modulate the secretion of proteolytic enzymes responsible for extracellular matrix degradation by tumour cells, when the neoplastic cells but not the non-neoplastic cells grown on FN/TN substrate showed positive immunofluorescence for collagenase. FN, LN or TN alone did not induce collagenase in the tumour cells. If the same is true in vivo, although a number of factors and interactions may implicate the ultimate outcome, the enhanced expression of TN in neoplastic lesions may have potential implications for tumour growth, differentiation, cellular adhesion, invasion and metastasis.

ヒトＨＳＰ９０アイソフォーム 特異的モクノローナル抗体の作製とヒト顎下腺腺癌細胞における応用

ヒトＨＳＰ９０アイソフォーム 特異的モクノローナル抗体の作製とヒト顎下腺腺癌細胞における応用

Identification of EGF as an angiogenic factor present in conditioned medium from human salivary gland adenocarcinoma cell clones with varying degrees of metastatic potential

We have previously shown that conditioned medium (CM) from metastasizing human salivary gland adenocarcinoma cell clones contains factor(s) that stimulate the proliferation and migration of bovine aortic endothelial (BAE) cells, and inhibit the production of collagenases by BAE cells (Azuma M. et al. (1993) Cancer Lett., 73, 85–93). To further characterize this, we evaluated the expression level of epidermal growth factor (EGF) secreted by a non-metastasizing cell clone (HSGc) and its metastasizing cell clones, and analysed the effect of EGF on the biologic behaviors of BAE cells. When the secretion of EGF by cell clones was estimated by enzyme-linked immunosorbent assay, metastasizing cell clones released a large amount of EGF as compared with HSGc. However, the number of EGF receptor was detected consistently at a level that was similar in all cell clones. With regard to the effect of EGF on the malignant potential of cell clones such as proteolytic aggressiveness, EGF did not affect the secretion of both collagenases and their inhibitor from cell clones. Alternatively, exogenous EGF stimulated the proliferation and migration of BAE cells, and inhibited the secretion of collagenases from BAE cells. Neutralization with a neutralizing antibody of EGF released into CM abolished the inhibitory effect of CM on the secretion of collagenases from BAE cells. Thus, the CM-contained factor, which is responsible for the induction of biologic behaviors of BAE cells, can be attributed to EGF.

Effects of media conditioned by a non-metastasizing human salivary gland adenocarcinoma cell clone and metastasizing clones from salivary gland and various other tissues on the proliferation, migration and protease production of bovine aortic endothelial cells in vitro

We demonstrate the role in tumor-associated angiogenesis of factors released into conditioned medium (CM) from in vitro human salivary gland cell clones with biological phenotypes ranging from non-metastasizing to metastasizing. A non-metastasizing human salivary gland adenocarcinoma cell clone HSGc and its subclone with metastatic potential (Gc2-100 cl-1) were employed. We also used metastasizing cell clones obtained by explant cultures of organs of Gc2-100 cl-1 tumor-bearing nude mouse. The proliferation and migration of bovine aortic endothelial (BAE) cells were significantly stimulated by the addition of CM obtained from Gc2-100 cl-1 and metastasizing cell clones, while CM from HSGc was ineffective. When the effect on protease secretion by BAE cells was examined, CM from Gc2-100 cl-1 and metastasizing cell clones inhibited the secretion of type IV collagenases by BAE cells much more than did CM from HSGc. These findings, therefore, may imply that Gc2-100 cl-1 and metastasizing cell clones secrete angiogenic factors that stimulate not only the proliferation and migration of endothelial cells but also the formation of basement membrane components necessary for the reconstruction of new blood vessels at migrated sites of endothelial cells by preventing the degradation of basement membrane component, type IV collagen.