KATO-III Gastric Cancer Cells Research Articles

In vitro cytotoxic activity on KATO-III cancer cell lines of mangiferolic acid purified from Thai Tetragonula laeviceps propolis

Gastric cancer is a global health concern, but current treatment with chemotherapy and surgery is often inadequate, prompting the exploration of alternative treatments. Propolis is a natural substance collected by bees known for its diverse properties linked to floral sources. The Dichloromethane Partitioned Extract (DPE) from Tetragonula laeviceps propolis, in Bankha district, Thailand was previously shown to possess significant cytotoxicity against KATO-III gastric cancer cells, while showing lower cytotoxicity toward WI-38 normal fibroblast cells. Here, the DPE was further fractionated by column chromatography, identified active fractions, and subjected to structural analysis using nuclear magnetic resonance spectroscopy. Cytotoxicity against KATO-III cells was reevaluated, and programmed cell death was analyzed using flow cytometry. Expression levels of cancer-related genes were measured using quantitative real-time reverse transcriptase PCR. Cardol C15:2 (compound 1) and mangiferolic acid (MF; compound 2) were discovered in the most active fractions following structural analysis. MF exhibited strong cytotoxicity against KATO-III cells (IC50 of 4.78–16.02 μg/mL), although this was less effective than doxorubicin (IC50 of 0.56–1.55 μg/mL). Morphological changes, including decreased cell density and increased debris, were observed in KATO-III cells treated with 30 μg/mL of MF. Significant induction of late-stage apoptosis and necrosis, particularly at 48 and 72 h, suggested potential DNA damage and cell cycle arrest, evidenced by an increased proportion of sub-G1 and S-phase cells. Doxorubicin, the positive control, triggered late apoptosis but caused more necrosis after 72 h. Furthermore, MF at 30 μg/mL significantly increased the expression level of COX2 and NFκB genes linked to inflammation and cell death pathways. This upregulation was consistent at later time points (48 and 72 h) and was accompanied by increased expression of CASP3 and CASP7 genes. These findings suggest MF effectively induces cell death in KATO-III cells through late apoptosis and necrosis, potentially mediated by upregulated inflammation-related genes.

Abstract B092: Intrinsic genomic plasticity of extrachromosomal DNA (ecDNA) enables oncogene amplified tumor cells to develop rapid acquired resistance to targeted therapy

Abstract Background: Focal high copy number oncogene amplification frequently occurs on extrachromosomal DNA (ecDNA), acentric circular DNA derived from chromosomes during neoplastic transformation and tumor progression. The unique properties of ecDNA facilitate a high degree of genomic plasticity and tumor heterogeneity, which enables rapid acquired resistance to targeted therapies and results in poor patient outcomes. Reintegrated ecDNA, also referred to as homogeneously staining regions (HSR), can exist in dynamic equilibrium with their circular form, although their potential adaptive and resistance properties have not been well characterized. Elucidating the genomic plasticity and mechanisms afforded to cancer cells harboring HSR may provide insights into new therapeutic approaches necessary to address this ecDNA-derived focal amplification. Methods: A novel model was developed that recapitulates the genomic plasticity of focal oncogene amplifications involving FGFR2 and EGFR in response to the FGFR inhibitor, infigratinib. Results: KATOIII gastric cancer cells harbor focal amplification of the FGFR2 oncogene (absolute copy number (CN) = 136 based on next-generation sequencing (NGS) that resides on an HSR as determined by FISH. Short- and long-read NGS revealed prior DNA breakpoints consistent with circular structures containing FGFR2, suggestive of reintegrated ecDNA as HSR. Despite initial sensitivity to FGFR2 inhibition (EC50 = 20 nM), cells acquired resistance following infigratinib dose escalation within 12 weeks. Infigratinib-resistant KATOIII cells retained the FGFR2 HSR while further amplifying FGFR2 on ecDNA, which correlated with increased FGFR2 CN, mRNA, and protein expression. Structural breakpoint analysis of the FGFR2 amplicon revealed conservation between baseline and ecDNA-amplified infigratinib resistant cells, suggesting that the HSR gave rise to ecDNA. Removal of infigratinib resulted in near-complete disappearance of FGFR2 ecDNA and return to near-baseline FGFR2 CN, protein expression, and signaling within 3 weeks. Strikingly, at high infigratinib concentrations that effectively inhibit FGFR2 signaling, cells rapidly switched to EGFR amplified on ecDNA, with concomitant reduction of FGFR2 ecDNA (CN = 553 to 238) and signaling and increased EGFR (CN = 3 to 30), mRNA, and EGFR signaling. Following ecDNA switching from FGFR2 to EGFR, KATOIII cells remained insensitive to infigratinib while acquiring sensitivity to the EGFR inhibitor erlotinib. Mutational analysis revealed no new oncogenic mutations including FGFR2 and EGFR, indicating the EGFR overexpression afforded by ecDNA was sufficient to drive acquired resistance to high infigratinib concentrations. Conclusions: These findings indicate that tumor cells with ecDNA-derived chromosomally reintegrated HSR retain the capacity to amplify ecDNA, thus enabling rapid adaptation to therapeutic pressure. Consequently, new treatment strategies that disable adaptive genomic plasticity are needed to address oncogene amplified cancers that leverage ecDNA biology. Citation Format: Kristen Turner, Homa Hemmati, Cory DuPai, John Bibay, Claire Klebs, Joan Chen, Debbie Liao, Christian Hassig, Shailaja Kasibhatla. Intrinsic genomic plasticity of extrachromosomal DNA (ecDNA) enables oncogene amplified tumor cells to develop rapid acquired resistance to targeted therapy [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B092.

TDP1 and TOP1 Modulation in Olaparib-Resistant Cancer Determines the Efficacy of Subsequent Chemotherapy

The aim of this study was to elucidate the carryover effect of olaparib to subsequent chemotherapy and its underlying mechanisms. We generated olaparib-resistant SNU-484, SNU-601, SNU-668, and KATO-III gastric cancer cell lines and confirmed their resistance by cell viability and colony forming assays. Notably, olaparib-resistant cell lines displayed cross-resistance to cisplatin except for KATO-III. Inversely, olaparib-resistant SNU-484, SNU-668, and KATO-III were more sensitive to irinotecan than their parental cells. However, sensitivity to paclitaxel remained unaltered. There were compensatory changes in the ATM/ATR axis and p-Chk1/2 protein expression. ERCC1 was also induced in olaparib-resistant SNU-484, SNU-601, and SNU-668, which showed cross-resistance to cisplatin. Olaparib-resistant cells showed tyrosyl-DNA phosphodiesterase 1 (TDP1) downregulation with higher topoisomerase 1 (TOP1) activity, which is a target of irinotecan. These changes of TOP1 and TDP1 in olaparib-resistant cells was confirmed as the underlying mechanism for increased irinotecan sensitivity through manipulated gene expression of TOP1 and TDP1 by specific plasmid transfection and siRNA. The patient-derived xenograft model established from the patient who acquired resistance to olaparib with BRCA2 mutation showed increased sensitivity in irinotecan. In conclusion, the carryover effects of olaparib to improve antitumor effect of subsequent irinotecan were demonstrated. These effects should be considered when determining the subsequent therapy with olaparib.

Effects of bone marrow-derived mesenchymal stem cells on invasion and metastasis of gastric cancer cells and its mechanism

Objective To investigate the role of bone marrow mesenchymal stem cells (BM-MSCs) in the invasion and metastasis of gastric cancer cells and to explore its mechanism. Methods SGC7901 and KATO-Ⅲ gastric cancer cells were co-cultured with BM-MSCs respectively, and the invasion ability of SGC7901 and KATO-Ⅲ gastric cancer cells were detected by Transwell assay. Secondly, CD133+ and CD133- cells were sorted from KATO-Ⅲ gastric cancers and co-cultured with BM-MSCs respectively to compare their changes in invasiveness. Meanwhile, the expressions of p-AKT and epithelial-mesenchymal transition(EMT) relative factors in gastric cancer cells were detected by Western-blot. The role of CD133 in BM-MSCs affecting the ability of invasion of gastric cancer cells was further vertified by the overexpression of CD133 in SGC7901 cells. SPSS17.0 software was used for statistical processing, and the stand deviation of the measurement data were expressed as the standard deviation, independent sample t test was conducted. Results The invasiveness of co-cultured SGC7901 and KATO-Ⅲ cells was significantly enhanced. The invasive ability of KATO-Ⅲ CD133+ cells co-cultured with BM-MSCs tended to increase more significantly than that of co-cultured CD133- cells[(259.0±24.0)vs(58.0±5.6), P<0.001]. The expressions of p-AKT, Snail and N-cadherin were significantly increased in co-cultured CD133+ cells (P=0.003, P=0.003, P=0.002), while the expression of E-cadherin was reduced (P=0.021). After co-cultured with BM-MSCs, the expression of E-cadherin was also reduced in CD133- cells (P=0.005), but the expressions of p-AKT, Snail and N-cadherin were no significantly changes (P=0.744, P=0.277, P=0.295). SGC7901 co-cultured with BM-MSC after overexpression of CD133 showed higher invasiveness than blank control group[(239.3±24.0) vs (103.3±15.5), P<0.001]. The expressions of p-AKT, Snail and N-cadherin were significantly increased when co-cultured with BM-MSCs in the group of CD133 overexpression (P=0.001, P=0.001, P=0.001), while the expression of E-cadherin was significantly decreased(P=0.003). The expressions of Snail and N-cadherin were also significantly increased after co-cultured with BM-MSCs in the blank control group (P=0.001, P=0.004), and the expression of E-cadherin was significantly decreased (P=0.018), while the expression of p-AKT was not significantly changed (P=0.193). Conclusions BM-MSCs can enhance the invasion and metastasis of gastric cancer cells by promoting the EMT of gastric cancer cells. CD133 may be involved in the regulation of EMT in gastric cancer cells through the PI3K/AKT signaling pathway. Key words: Stomach neoplasms; Mesenchymal stem cells; Bone marrow; Epithelial-mesenchymal transition; CD133

Overexpression of miR-17 in gastric cancer is correlated with proliferation-associated oncogene amplification.

The molecular mechanism underlying microRNA (miR)-17 overexpression has not been clearly evaluated in gastric cancer. We aimed to evaluate the functional roles of miR-17 in gastric cancer and test its viability as a therapeutic target. We conducted comparative genomic hybridization and expression array analyses on human gastric cancer tissue samples, as well as evaluating the functional roles of miR-17 in gastric cancer cell lines and transgenic mice. miR-17 overexpression in gastric cancer patients was associated with copy number gain of proliferation-associated oncogenes such as MYC, CCNE1, ERBB2, and FGFR2. Copy number gain of MIR17HG gene (13q31.3) was rare, with an overall frequency of 2% in gastric cancers (1 of 51). miR-17 knockdown suppressed the monolayer and anchorage-independent growth of FGFR2-amplified KATO-III gastric cancer cells. mir-17-92 TG/TG mice overexpressing the mir-17-92 cluster under the villin promoter developed spontaneous benign tumors in the intestinal tract (log-rank P for tumor-free survival = 0.069). Taken together, miR-17 overexpression in gastric cancer was rarely associated with MIR17HG gene amplification, but correlated with proliferation-associated oncogene amplification. Therefore, miR-17-targeting approach may benefit patients with gastric cancers harboring proliferation-associated oncogene amplification.

Role of p53β in the inhibition of proliferation of gastric cancer cells expressing wild-type or mutated p53.

p53 is a tumor suppressor gene whose mutation is highly associated with tumorigenesis. The present study investigated the role of p53β in the inhibition of proliferation of gastric cancer cell lines expressing wild-type or mutated p53. Wild-type p53 is expressed in MKN45 cells, but deleted in KATOIII cells, whereas mutated p53 is expressed in SGC7901 cells. The mRNA expression levels of p53β and Δ133p53 were detected in MKN45, SGC-7901 and KATOIII gastric cancer cell lines using nested polymerase chain reaction (PCR). The mRNA expression levels of p53, p53β and B-cell lymphoma 2-associated X protein (Bax) were detected in the MKN45 and SGC-7901 cells following treatment with cisplatin by reverse transcription-PCR. The inhibition of cellular proliferation following treatment with cisplatin was measured by MTT assay. The results of the present study demonstrated that both p53β and Δ133p53 mRNA were expressed in the MKN45 cells, whereas only p53β mRNA was expressed in the SGC7901 cells. No expression of p53β or Δ133p53 mRNA was detected in the KATOIII cells. Following treatment with cisplatin, the number of both MKN45 and SGC-7901 cells was significantly reduced (P<0.001). In the MKN45 cells, p53β, p53 and Bax mRNA expression levels gradually increased with the dose of cisplatin, and the expression of p53β was positively correlated with the expression of p53 (tr=6.358, P<0.05) and Bax (tr=8.023, P<0.05). In the SGC-7901 cells, the expression levels of p53β, p53 and Bax mRNA did not alter with the dose of cisplatin, and the expression of p53β was positively correlated to the expression of p53 (tr=26.41, P<0.01) but not that of Bax. The present study identified the different roles of the p53β isoform in gastric cancer cells with different p53 backgrounds. Enhanced knowledge regarding the p53 status is required for the development of specific biological therapies against gastric cancer.

Abstract B81: AUY922, a heat shock protein 90 inhibitor, actively inhibits human gastric cancer cells via down regulation of growth factor receptors, and acts synergistically with relevant chemotherapeutic agents

Abstract Heat shock protein 90 (Hsp90) is the most abundant cellular chaperone and has nearly 100 pivotal client proteins, which are commonly dysregulated in cancers. The positive rate of Hsp90beta in gastric cancer tissue was approximately 30%, and was higher than non-cancerous gastric mucosa. Hsp90 inhibitors should have great potentials in the exploration for treatment strategy in gastric cancer (GC). AUY922, kindly provided by Novartis Pharma AG, is a novel resorcinylic isoxazole amide Hsp90 inhibitor. A panel of human GC cell lines (N87, AGS, SNU-1, SNU-16, KATO-III from ATCC) was used in this study. Using 72-hour MTT colorimetric assay, we demonstrated that AUY922 has highly active growth inhibitory effects as a single agent of IC50 in the ranges between 8.1 ± 0.2 and 14.7 ± 0.8 nM (except the c-met-overexpressing KATO-III cells with a IC50 of 158.1 ± 5.1 nM), which is more active than 17-AAG (17-allylamino-17-demethoxygeldanamycin) and 17-DMAG (17-dimethylaminoethyl-amino-17-demethoxygeldanamycin) of IC50 between 17.2 ± 0.6 and 61.3 ± 1.9 nM, and IC50 between 10.5 ± 0.5 and 24.3 ± 1.0 nM, respectively. By MTT colorimetric assay and median effect analysis, AUY922 has significant synergistic cytotoxicity with cisplatin, oxaliplatin, 5-fluorouracil (5-FU), and irinotecan, while acts antagonistically with paclitaxel and docetaxel in GC cells. Several important growth factor receptors are highly expressed in GC, namely 50-60% of EGFR (epidermal growth factor receptor, HER1), 15–22% of HER-2/neu, and 32–58% of c-met (hepatocyte growth factor receptor, HGF receptor), respectively. By immunoblotting analysis, AUY922 effectively down-regulates growth factor receptors (c-met, EGFR, and HER-2/neu) with a concentration of 25nM, 25nM, and 125nM in KATO-III GC cells, respectively. The c-met-overexpressing KATO-III cells are highly resistant to AUY922. In KATO-III cells, knocking-down of c-met by siRNA, then examining by 48-hour MTT colorimetric assay may partly abrogate the AUY922 resistance and increase sensitivity to AUY922 by decreasing IC50 from 790.0 ± 10.6 nM to 350.8 ± 5.4 nM. Our data indicate that AUY922 actively inhibits human GC cells via down regulation of growth factor receptors, and acts synergistically with various relevant chemotherapeutic agents. It is reasonable to use AUY922 alone, or to combine AUY922 with relevant chemotherapeutic agents (cisplatin, oxaliplatin, 5-FU, or irinotecan) in future development of clinical trials for gastric cancers. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B81.

Immune response of heparinase gene modified dendritic cell-based vaccine on gastric cancer cells

To explore the feasibility of heparinase vaccine in active immunity for gastric cancer. Dendritic cells originated from the peripheral blood mononuclear cells (PBMC) of healthy HLA-A2 positive donors were transfected with recombinant adenovirus containing heparinase full length cDNA of heparanase to generate heparanase gene modified DC vaccine. T lymphocytes from the same donors were activated by those genetically modified DC vaccine repeatedly to generate heparanase specific cytotoxicity T lymphocytes (CTL). CTL-mediated cell lysis to gastric cancer cells of the lines KATO-III and SGC-7901 was analyzed in vitro by standard (51)Cr releasing assay. Heparinase specific CTL were co-cultured with KATO-III and SGC-7901 cells, and then ELISA was used to detect the IFN-gamma release. Expression of heparanase was significantly increased in the DCs transfected with heparinase recombinant adenovirus. Heparanase specific CTL generated from the genetically modified DC vaccine exhibited potent lysis to the KATO-III gastric cancer cells positive in both heparinase and HLA-A2 at each E/T ratio, whereas, these heparinase specific CTL could not lyse the SGC-7901 cells positive to heparinase but negative to HLA-A2, with a specific lysis rate of only 11.1% +/- 4.6% even at an E:T ratio of 40:1. Further study showed that heparanase vaccination had no detectable lysis on the autologous lymphocytes in vitro with a specific lysis rate of only 11.4% +/- 7.9% even at an E:T ratio of 40:1. The IFN-gamma release amount when the heparanase specific CTL were co-cultured with the KATO-III cells was 280.4 pg/ml +/- 23.5 pg/ml, significantly higher than that when the heparanase specific CTL were co-cultured with the rAd5-Lacz modified DC (120.6 pg/ml +/- 18.9 pg/ml), and that of the IL-2 stimulated T cells (60.0 pg/ml +/- 10.6 pg/ml, both P < 0.05). In contrast the IFN-gamma release amounts of the SGC-7901 cells and autologous lymphocytes remained unchanged when they were co-cultured with either above-mentioned effector cells (both P > 0.05). DC genetically modified by heparanase gene activate heparanase specific CTL and induce potent immune response against HLA-matched and heparinase positive gastric cancer cells in vitro, whereas they have no killing effect on autologous lymphocytes. Heparanase is an effective and safe target for immunogen therapy of tumor, thus providing a new biotherapy method for advanced gastric cancer.

Expression of Endomembrane Calcium Pumps in Colon and Gastric Cancer Cells: INDUCTION OF SERCA3 EXPRESSION DURING DIFFERENTIATION

Calcium mobilization from the endoplasmic reticulum (ER) into the cytosol is a key component of several signaling networks controlling tumor cell growth, differentiation, or apoptosis. Sarco/endoplasmic reticulum calcium transport ATPases (SERCA-type calcium pumps), enzymes that accumulate calcium in the ER, play an important role in these phenomena. We report that SERCA3 expression is significantly reduced or lost in colon carcinomas when compared with normal colonic epithelial cells, which express this enzyme at a high level. To study the involvement of SERCA enzymes in differentiation, in this work differentiation of colon and gastric cancer cell lines was initiated, and the change in the expression of SERCA isoenzymes as well as intracellular calcium levels were investigated. Treatment of the tumor cells with butyrate or other established differentiation inducing agents resulted in a marked and specific induction of the expression of SERCA3, whereas the expression of the ubiquitous SERCA2 enzymes did not change significantly or was reduced. A similar marked increase in SERCA3 expression was found during spontaneous differentiation of post-confluent Caco-2 cells, and this closely correlated with the induction of other known markers of differentiation. Analysis of the expression of the SERCA3 alternative splice isoforms revealed induction of all three known iso-SERCA3 variants (3a, 3b, and 3c). Butyrate treatment of the KATO-III gastric cancer cells led to higher resting cytosolic calcium concentrations and, in accordance with the lower calcium affinity of SERCA3, to diminished ER calcium content. These data taken together indicate a defect in SERCA3 expression in colon cancers as compared with normal colonic epithelium, show that the calcium homeostasis of the endoplasmic reticulum may be remodeled during cellular differentiation, and indicate that SERCA3 constitutes an interesting new differentiation marker that may prove useful for the analysis of the phenotype of gastrointestinal adenocarcinomas.

Bax overexpression enhances cytochrome c release from mitochondria and sensitizes KATOIII gastric cancer cells to chemotherapeutic agent-induced apoptosis.

To evaluate whether overexpression of Bax, an apoptosis-promoting gene, sensitizes KATOIII gastric cancer cells to apoptosis induced by chemotherapeutic agents, three stable cell lines of KATOIII transfected with Bax (KATOIII-Bax), Bcl-2 (KATOIII-Bcl-2), or control pCI-neo expression vector (KATOIII-pCI-neo) were established. The cells were treated with paclitaxel, 5-fluorouracil, or doxorubicin, and the apoptotic response was measured. Our results showed that the sensitivity of the KATOIII-Bax cells to chemotherapeutic agents was enhanced compared with that of the KATOIII-pCI-neo cells, and the KATOIII-Bcl-2 cells were more resistant to these agents. Western blotting revealed that cytochrome c level in the cytosol fraction of the KATOIII-Bax cells was higher than that of the KATOIII-pCI-neo cells. Significant increase of cytochrome c level in the cytosol fraction of the KATOIII-Bax cells was detected 24 h after exposure to chemotherapeutic agents, when apoptotic cells were less than 10%. The cytochrome c level in the cytosol fraction of the KATOIII-Bax cells was higher than that of the KATOIII-pCI-neo cells at all time points examined after exposure to chemotherapeutic agents. Marked activation of caspase-3 in the KATOIII-Bax cells was observed 48 h and 72 h after exposure to chemotherapeutic agents compared with that in the KATOIII-pCI-neo cells. Consistently, zVAD-fmk, a pancaspase inhibitor, repressed the paclitaxel-induced apoptosis. In addition, Bcl-2 overexpression strongly blocked KATOIII cell apoptosis by inhibiting the cytochrome c release from mitochondria and caspase-3 activation. These findings suggest that cytochrome c release is a major mechanism of apoptotic response and Bax overexpression sensitizes KATOIII cells to chemotherapeutic agent-induced apoptosis through enhancing the release of cytochrome c from mitochondria.

Frequent expression of complement resistance factors CD46, CD55, and CD59 on gastrointestinal cancer cells limits the therapeutic potential of monoclonal antibody 17-1A.

One reason for the failure of monoclonal antibody (mab) trials in most cancer patients might be the presence of complement resistance factors that inhibit complement dependent cytotoxicity (CDC) and the release of inflammatory mediators (e.g., anaphylatoxins). We have determined the expression of CD46, CD55, and CD59 in five gastric, three colon, and seven pancreatic human cancer cell lines by immunostaining. The complement activating properties of mabs and conjugates with cobra venom factor (CVF) were studied in a 51Cr-release toxicity assay and in an ELISA to determine the release of C3a. Virtually all tumor cell lines strongly expressed CD46, CD55, and CD59, except KATOIII gastric cancer cells (CD55 and CD59 negative). In accordance with other studies we could confirm that expression of CD55 and CD59 inhibits a complement activation by mabs. Whereas 17-1A was able to induce a cytotoxic complement activation on KATOIII cells, neither a CDC nor an anaphylatoxin release (C3a) was observed on MKN28 cells (strong expression of CD55 and CD59). Conjugation with CVF, a strong activator of the alternative pathway of complement, could partially restore the complement activation by mabs. A 17-1A-CVF conjugate, although still nontoxic, induced the release of the anaphylatoxin C3a on both cell lines. The same observations were made in PancTuI pancreatic cancer cells treated with a conjugate of the mab CA19-9 and CVF. Our study shows that complement resistance is a frequent event in gastrointestinal cancer, limiting the potential of monoclonal antibodies. Mabs, when conjugated with CVF, partially retain complement activating properties by releasing C3a, which in vivo will support a cellular immune response.

A Soluble Form of K-sam FGFR2 Protein in the Culture Medium of Human Gastric Cancer Cells

K-SAM/FGFR2 gene encodes a receptor tyrosine kinase which belongs to the fibroblast growth factor receptor family and is amplified and overexpressed in KATO-III gastric cancer cells. To characterize K-sam proteins in cancer cells, anti-K-sam rabbit polyclonal antibody PK1-2 was raised and used for the immunoprecipitation analysis. 135, 125, and 110-kDa transmembrane proteins were detected in KATO-III cell lysate, while a soluble truncated 85-kDa K-sam protein was found in the conditioned medium. The molecular size of the soluble K-sam protein does not match with those predicted from the secreted forms of the K-sam cDNA which have been cloned so far. The soluble K-sam protein was highly N-glycosylated like the transmembrane versions, and N-glycosylation appeared to be necessary for its release.