Quantitative cell signalling analysis reveals down‐regulation of MAPK pathway activation in colorectal cancer

Mitogen‐activated protein kinases (MAPK) are considered to play significant roles in colonic carcinogenesis and kinase inhibitor therapy has been proposed as a potential tool in the treatment of this disease. Reverse‐phase microarray assays using phospho‐specific antibodies can directly measure levels of phosphorylated protein isoforms. In the current study, samples from 35 cases of untreated colorectal cancer colectomies were laser capture‐microdissected to isolate epithelium and stroma from cancer as well as normal (i.e. uninvolved) mucosa. Lysates generated from these four tissue types were spotted onto reverse‐phase protein microarrays and probed with a panel of antibodies to ERK, p‐ERK, p38, p‐p38, p‐JNK, MEK and p‐MEK. Whereas total protein levels were unchanged, or slightly elevated (p38, p = 0.0025) in cancers, activated isoforms, including p‐ERK, p‐p38 and p‐JNK, were decreased two‐ to four‐fold in cancers compared with uninvolved mucosa (p < 0.0023 in all cases except for p‐JNK in epithelium, where decrement was non‐significant). This was backed up by western blotting. Dukes' stage B and C cancers displayed lower p‐ERK and p‐p38 expression than Dukes' stage A cancers, although this was not statistically significant. It is concluded that MAPK activity may be down‐regulated in colorectal cancer and that further exploration of inhibitory therapy in this system should be carefully evaluated if this finding is confirmed in larger series. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Protective mechanism of kaempferol against Aβ25-35-mediated apoptosis of pheochromocytoma (PC-12) cells through the ER/ERK/MAPK signalling pathway

IntroductionProgressive accumulation of amyloid-β (Aβ) is a pathological trait of Alzheimer’s disease (AD). Amyloid-β increases free radical production in neuronal cells, leading to neuronal cell death. Hormone replacement therapy can reduce the incidence of AD, and oestrogen significantly improves the clinical signs in patients with AD. However, the long-term use of oestrogen causes a variety of diseases. Phytoestrogens have been reported to bind and activate oestrogen receptors in mammals and humans to produce oestrogen-like or anti-oestrogen-like effects. Kaempferol is a flavonoid phytoestrogen that can produce a certain protective effect in neurons. However, the molecular mechanism of kaempferol in AD is unclear.Material and methodsThis study used pheochromocytoma (PC-12) cells that were damaged by Aβ_25–35 as an in vitro model of AD, and oestradiol was a positive control. The cells were incubated with kaempferol alone or in combination with fulvestrant (an antagonist of ER) and U0126 (an inhibitor of ERK) in Aβ_25–35 culture. Cell activity was measured by the MTT method. Cell apoptosis was evaluated by flow cytometry. Gene and protein expression levels were tested by qRT-PCR and Western blotting.ResultsThis study demonstrated that kaempferol protected PC-12 cells from Aβ_25–35-induced cell death and apoptosis in a dose-dependent manner. Treatment with fulvestrant (an antagonist of ER) and U0126 (an inhibitor of ERK) significantly increased the apoptosis of PC-12 cells. Moreover, kaempferol promoted the expression of anti-apoptotic molecules and inhibited the expression of pro-apoptotic molecules, which were blocked by fulvestrant and U0126.ConclusionsKaempferol protected PC-12 cells against Aβ_25–35-induced cell apoptosis through the ER/ERK/MAPK signalling pathway.     

Age-dependent loss of NGF signaling in the rat basal forebrain is due to disrupted MAPK activation

The loss of nerve growth factor (NGF) and its high affinity receptor TrkA has been implicated in the loss of cholinergic tone and function in Alzheimer's disease (AD) and normal aging. We employed an animal model of aging, the aged rat, which also exhibits memory loss and NGF alterations. Basal forebrain TrkA levels increased after injection of NGF in the hippocampus within 1h in young rats, but this response was diminished in aged animals as determined by Western blot analysis. Further, NGF activated MAPK pathways without changing total ERK levels and the activation of these pathways was also diminished in aged animals. The exogenous NGF injection did not appear to activate the PI-3K pathway or alter total levels of Akt significantly. These data shed light on mechanisms of NGF signaling in the CNS, and alterations in this signaling cascade associated with age and memory loss. These findings might lead to development of novel treatment therapies for the memory loss associated with AD and other age-associated neurodegenerative diseases.     

A polymorphism in a phosphotyrosine signalling motif of CD229 (Ly9, SLAMF3) alters SH2 domain binding and T‐cell activation

Signalling lymphocyte activation molecule (SLAM) family members regulate activation and inhibition in the innate and adaptive immune systems. Genome‐wide association studies identified their genetic locus (1q23) as highly polymorphic and associated with susceptibility to systemic lupus erythematosus (SLE). Here we show that the Val₆₀₂ variant of the non‐synonymous single nucleotide polymorphism (SNP) rs509749 in the SLAM family member CD229 (Ly9, SLAMF3) has a two‐fold lower affinity compared with the SLE‐associated Met₆₀₂ variant for the small adaptor protein SAP. Comparison of the two variants in T‐cell lines revealed the Val₆₀₂ variant to be significantly more highly expressed than CD229 Met₆₀₂. Activation was diminished in cells expressing CD229 Val₆₀₂ compared with CD229 Met₆₀₂ as measured by up‐regulation of CD69. There was no correlation between homozygosity at rs509749 and activation in peripheral blood mononuclear cells from healthy donors. These findings identify potential mechanisms by which a single SNP can perturb fine‐tuning in the immune system with significant functional consequences.     

Knockdown of apoptosis repressor with caspase recruitment domain (ARC) increases the sensitivity of human glioma cell line U251MG to VM-26

Previous studies have demonstrated that apoptosis repressor with caspase recruitment domain (ARC) is up-regulated in many forms of malignant tumors and low levels of ARC protein were expressed in normal human brain tissue. Little is known expression of ARC in glioma. Here, we found that ARC protein was highly expressed in primary human glioma when compared with normal brain tissues. A decrease in cell viability and an increase in apoptosis were observed in U251MG cells after ARC was knocked down. Knockdown of ARC was confirmed by western blotting. Knockdown of ARC promoted caspase-8, caspase-3 activation and Bax accumulation. These results indicate that ARC has a anti-apoptosis function in glioma.     

Characterization of a newly established human acinic cell adenocarcinoma cell line (HACC) originating from the salivary gland: Morphological features and role of various growth factors on the growth of the HACC cell line

Human acinic cell adenocarcinoma cell (HACC) line was established from the pleural effusion that contains meta-static tumor cells of acinic cell adenocarcinoma of papillary and microcystic type originating from the parotid gland. The HACC cells grew in an adherent monolayer with a doubling time of 66 h. Implanted tumor of SCID mice revealed similar histologlcal findings to that of the primary tumor. The HACC cells produced mucin and expressed epithelial markers as well as α₁-antitrypsin and lysozyme, whereas salivary peptide P-C was expressed in cultured HACC cells but not In the primary and Implanted HACC cell tumors. S-100 protein was also expressed in both the primary tumor and HACC cell line. Neither amplification of common oncogenes nor expression of p53 was observed. The receptor for epidermal growth factor (EGF) was expressed, indicating EGF and transforming growth factor-α (TGF-α) enhanced the growth of the HACC line. Unexpectedly, tumor necrosis factor-α (TNF-α) also enhanced the growth of the HACC line significantly. However, there was no evidence of autocrine growth using these growth factors. In contrast, TGF-β1 inhibited the growth of the HACC cell line through apoptosis. The HACC cell line has features similar to both acinar and intercalated ductal cells of the salivary gland. Epidermal growth factor, TGF-α and TNF-α are potential growth factors for the HACC cell line. The HACC cell line may be a good model for studying the biological behavior of salivary gland neoplasms.     

原发性食管腺癌癌基因、抗癌基因的扩增与其蛋白产物的对比研究

采用免疫组化和原位分子杂交技术观察ｐ５３、Ｈ－ｒａｓ、ｃ－ｅｒｂＢ－２、ｃ－ｍｙｃ癌基因和抗癌基因及其蛋白产物在原发性食管腺癌（ＰＥＡ）中的联合表达情况，以探讨ＰＥＡ的发生发展机制。结果表明，ＰＥＡ中的ｐ５３表达较食管鳞癌高；ｃ－ｅｒｂＢ－２变化可能是ＰＥＡ癌变的重要分子学改变；ＰＥＡ中Ｈ－ｒａｓ与ｃ－ｍｙｃ都有基因扩增蛋白产物的过表达，显示两者在ＰＥＡ的癌变过程中有协同作用；ｐ５３、ｃ－ｅｒｂＢ－２的表达与ＰＥＡ的分化程度呈负相关，即分化程度越差，阳性率越高；ＰＥＡ中癌基因和抗癌基因在基因水平及其蛋白产物表达上的改变基本一致。     

Differential regulation of mast cell cytokines by both dexamethasone and the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580

Activated mast cells generate multiple cytokines but it is not known if these can be differentially regulated by pharmacological agents. We report here that the glucocorticoid dexamethasone (DEX) preferentially inhibited Ag-induced expression of IL-4 and IL-6 mRNA relative to TNF-alpha mRNA in RBL-2H3 cells. Likewise, the drug more readily inhibited release of IL-4 than TNF-alpha protein. SB203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK), enhanced Ag-induced TNF-alpha mRNA expression without affecting IL-4 or IL-6 mRNA. At the protein level, SB203580 exerted little effect on TNF-alpha release but inhibited IL-4 release; notably, the ratio of TNF-alpha : IL-4 increased markedly with the concentration of SB203580, confirming the differential regulation of these cytokines. PD98059, an inhibitor of MAPK kinase (MEK), a component of the p44/42 MAPK pathway, partially inhibited Ag-induced expression of mRNA for all three cytokines while cyclosporin A inhibited Ag-induced IL-4 and IL-6 mRNA more readily than TNF-alpha mRNA. Ag activation of the cells led to phosphorylation of p38 and p44/42 MAPK but this was not influenced by DEX. In conclusion, mast cell cytokines can be differentially regulated pre- and post-translationally by DEX and SB203580 but there does not appear to be a direct mechanistic link between the actions of these two drugs.     

Clonal analysis of a human lymphoblastoid cell line (B17) secreting antibody to N-acetyl-D-glucosamine

In this paper we analyse the clonal composition of a human lymphoblastoid B-cell line secreting IgM/k antibody to N-acetyl-D-glucosamine, the immunodominant sugar of Group-A-streptococcal carbohydrate. Besides non-antibody secreting cells, the line consists of two clonotypes of antibody-secreting cells: B17 cells producing over 90% and F6 cells producing less than 10% of the antibody in the supernatant. The proportions of B17 and F6 cells in the cell line seem to be similar to the proportion of antibodies in the supernatant. F6 cells can be isolated by cloning and maintained as stable lines, whereas this is more difficult with B17 cells. The results suggest that upon establishment of the line, at least two N-acetyl-D-glucosamine-specific B cells were immortalized and coexist together as independent clonotypes. Although F6 cells seem to have a slight tissue culture advantage, they represent the minor clonotype in the B17 cell line.     

The prognostic importance of the nucleolar organizer region (AgNOR), Ki-67 and proliferating cell nuclear antigen (PCNA) in primary nonurachal bladder adenocarcinoma

The aim of this study was to investigate the role of tumor proliferation in patients with nonurachal bladder adenocarcinoma. Samples were obtained from 16 patients (12 men and 4 women, mean age 62 years) with primary nonrurachal bladder adenocarcinoma. The 16 formalin-fixed specimens were stained immunohistochemically for Ki-67 antigen and PCNA using MIB-1 and PC-10 antibodies. In addition, the AgNOR quantity was assessed using the colloid silver nitrate staining technique in all cases. The Ki-67, PCNA and AgNOR proliferation indices were found to be significantly higher in high-grade and invasive tumors. The higher the grade (p<0.01) and stage (p<0.01), the higher were the proliferation indices. Patients whose tumor samples had a high Ki-67, PCNA and AgNOR proliferation index showed a higher incidence of local recurrence (p<0.01) and distant metastasis (p<0.01). In conclusion, our results suggest that Ki-67, PCNA and AgNOR proliferation scores may be important prognostic indices in nonurachal bladder adenocarcinomas.     

Mucin histochemistry in primary adenocarcinoma of the urinary bladder (of urachal or vesicular origin) and metastatic adenocarcinoma originating in the colorectum

In order to evaluate the mucin histochemistry of primary adenocarcinomas (PA) of the urinary bladder and metastatic adenocarcinoma (MA) originating in the colorectum, 52 PA and nine MA were examined. It was determined that the percentage of cases in which more than 25% of the tumor was stained by each of the following: (i) Alcian blue pH 2.5 periodic acid-Schiff (AB-PAS); (ii) high iron diamine-AB (HID-AB); (iii) periodic acid-sodium borohydride-potassium hydroxide-PAS (PA-SB-PH-PAS); (iv) galactose oxidase- Schiff (GOS); and (v) paradoxical concanavalin A stain (PCS). For PA, the values obtained were: 75% of cases (blue, AB-PAS), 85% (magenta, AB-PAS), 71% (black, HID-AB), 75% (blue, HID-AB), 0% (PA-SB-PH-PAS), 19% (GOS), 8% (class II concanavalin A (Con A)-reactive mucin)), and 0% (class III Con A-reactive mucin). For MA, the corresponding values were 33, 22, 0, 11, 0, 0, 11, and 0%, respectively. A higher percentage of PA than MA cases showed staining in AB-PAS for acidic and neutral mucins, in HID-AB for sialo- and sulfomucins, and in GOS for terminal beta-galactose and beta-N-acetylgalactosamine. PA and MA were significantly different in terms of both frequency of staining with AB-PAS and frequency of staining with HID-AB. However, the overlap was such that in practice, it might be difficult, if not impossible, to use mucin histochemistry to inform a differential diagnosis. In view of the differences in AB-PAS and HID-AB positivity between PA and MA, we speculate that MA (originating in the colorectum) may have undergone structural distortion affecting the production and/or secretion of neutral mucins and acidic mucins (sialo- and sulfomucins) during metastasis or invasion.     

Morphological analysis of the cellular interaction between thymocytes and a thymic stromal cell line (TEL-2)

In a previous study (Nakashima et al., Eur. J. Immunol., 20: 47-53, 1990), a cloned stromal cell line TEL-2 was established from Balb/c mouse thymus. Incubation of thymocytes with TEL-2 cells resulted in the selective elimination of CD4 and CD8 double-positive thymocytes from the culture. In the present report, both phase-contrast and scanning electron microscopes were used to examine, at various time intervals, TEL-2 cells cocultivated with thymocytes in order to elucidate the kinetic sequence of their cellular interaction. The thymocytes attached to the TEL-2 cell surface were more numerous at early times (30 min to 1 h), and their number decreased gradually with time. In contrast, the thymocytes that migrated into the TEL-2 cell layers were less abundant at early times, their number increasing with time thereafter. Destruction of the regular arrangement of TEL-2 cells was found at later than 1 h, suggesting active cellular interaction. The thymocytes adherent to the TEL-2 cell surface were found to be of various shapes and often showed variable profiles, e.g., extending small cytoplasmic processes along the surface of TEL-2 cells or appearing ameboidal. A remarkable feature of the TEL-2 cells was that they formed numerous "round spaces" at the surface of the TEL-2 cell layers. The thymocytes were often located around "round spaces," and some were seen migrating into TEL-2 cell layers through these round spaces. In addition, complementary examinations by transmission electron microscopy revealed that the internalization of thymocytes into TEL-2 cells occurs inside the TEL-2 cell layers after migration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative studies on the secretion and activation of MMPs in two reconstructed human skin models using HaCaT- and HaCaT-ras-transfected cell lines

Matrix metalloproteinases play an important role in tissue regeneration, wound healing and tumor invasion. Our previous studies have shown a higher motility of HaCaT-ras-transfected cells compared with HaCaT or normal human keratinocytes (NHK) in correlation with a higher secretion of MMP-2 (72 kDa) or MMP-9 (92 kDa), according to the medium used for cell cultures. Presently, the expression and activity of MMPs were investigated in two reconstructed skin models, using a dead de-epidermized dermis (DED) or a dermal substitute including living fibroblasts. In all experiments, MMP-9 was essentially secreted by NHK and to a greater extent by HaCaT cells. Its active form (86 kDa) was only detected in both reconstructed skin models according to keratinocyte differentiation. MMP-2 was mainly secreted by living fibroblasts included in the dermal substitute skin model. In this case, its activation was up-regulated when HaCaT cell lines were seeded onto the dermal substitute according to their culture at air/liquid interface as shown for MMP-9. The collagenase MMP-1 and stromelysin-1 (MMP-3), susceptible to activate pro-MMP-2 and -9, respectively, were detected in their inactive form by ELISA. MMP-1 was expressed in both models but MMP-3 required the presence of living fibroblasts. Their activities were not detected using specific fluorogenic substrates. In the skin equivalent model using HaCaT, the extensive secretion and activation of MMP-2 and MMP-9 could explain the defect observed in basal membrane reconstruction, suggesting a direct interaction of HaCaT with fibroblasts.     

Comparative analysis of the internalization of the macrophage receptor sialoadhesin in human and mouse primary macrophages and cell lines

Sialoadhesin (Sn) is a surface receptor expressed on resident macrophages with the ability to bind with sialic acids. During inflammation, an upregulation of Sn is observed. Upon binding of monoclonal antibodies to Sn, the receptor becomes internalized and this has been observed in multiple species. The latter characteristic, combined with the strong upregulation of Sn on inflammatory macrophages and the fact that Sn-positive macrophages contribute to certain inflammatory diseases, makes Sn an interesting entry portal for phenotype-modulating or cytotoxic drugs. Such drugs or toxins can be linked to Sn-specific antibodies which should enable their targeted uptake by macrophages. However, the activity of such drugs depends not only on their internalization but also on the intracellular trafficking and final fate in the endolysosomal system. Although information is available for porcine Sn, the detailed mechanisms of human and mouse Sn internalization and subsequent intracellular trafficking are currently unknown. To allow development of Sn-targeted therapies, differences across species and cellular background need to be characterized in more detail. In the current report, we show that internalization of human and mouse Sn is dynamin-dependent and clathrin-mediated, both in primary macrophages and CHO cell lines expressing a recombinant Sn. In primary macrophages, internalized Sn-specific F(ab’)₂ fragments are located mostly in the early endosomes. With Fc containing Sn-specific antibodies, there is a slight shift towards lysosomal localization in mouse macrophages, possibly because of an interaction with Fc receptors. Surprisingly, in CHO cell lines expressing Sn, there is a predominant lysosomal localization. Our results show that the mechanism of Sn internalization and intracellular trafficking is concurrent in the tested species. The cellular background in which Sn is expressed and the type of antibody used can affect the intracellular fate, which in turn can impact the activity of antibody-based therapeutic interventions via Sn.     

Failure of pertussis toxin to inhibit activation of guanylate cyclase by the heat-stable enterotoxin of Escherichia coli (STa) in the T84 cell line.

The heat-stable enterotoxin (STa) of Escherichia coli causes intestinal secretion by stimulating guanylate cyclase, an enzyme believed to be distinct from the STa receptor. Pertussis toxin (PT) has been reported to block the ability of STa to stimulate guanylate cyclase in rat intestinal mucosa (S. A. Epstein, R. A. Giannella, and H. J. Brandwein, FEBS Lett. 203:44-48, 1986). This suggested that a guanine nucleotide regulatory protein (G protein) coupled the STa receptor to guanylate cyclase, a function not previously recognized for G proteins. We sought to explore this phenomenon and, if possible, to identify this G protein. Initial experiments with the human colon carcinoma cell line T84 revealed that higher-than-expected concentrations (1 micrograms/ml) of PT were needed to intoxicate cells, as assessed by ADP-ribosylation of endogenous PT substrate, but that 99 to 100% intoxication could be achieved. Homogenates made from fully intoxicated cells did not differ from controls in basal or STa-stimulated guanylate cyclase activity, and cyclic GMP accumulation in intact T84 cells was not changed by PT treatment. We conclude that a PT-sensitive G protein is not involved in the stimulation of cyclic GMP production by the enterotoxin STa.     

Establishment and characterization of a human lung adenocarcinoma cell line (LC-2/ad) producing α1-antitrypsin in vitro

A new human cell line, LC-2/ad was established from pleural effusion of pulmonary adenocarcinoma of a 51 year old Japanese female. The LC-2/ad cells exhibit an epithelial appearance and a tendency to form small domes as observed with phase-contrast microscopy. The modal chromosome number was 53–56. Plating efficiency and doubling time were 6.8% and 58 h, respectively (32th passage). Immunocytochemlcally, the cells were strongly positive for CEA and cytokeratins including cytokeratin no. 18 which is present in simple epithella. Ultrastructurally, the cultured cells were characterized by well-formed junctional complexes and microvilli. Subcutaneous injection of 5 × 10⁶ cells into a nude mouse resulted in tumor formation classified histologically as a moderately differentiated adenocarcinoma. This cell line produced at least two functionally active trypsin inhibitors together with several proteinases in vitro. The main inhibitor was purified partially from the serum-free conditioned medium and confirmed immunologically as human α₁-antitrypsin (AAT). Immunohistochemically, the xenografted tumor was also positive for AAT. The cell line LC-2/ad is useful for the study of tumor-derived serine proteinase inhibitors, in particular AAT.     

Oncogenic levels of mitogen‐activated protein kinase (MAPK) signaling of the dinucleotide KRAS2 mutations G12F and GG12‐13VC

We previously reported the occurrence of novel dinucleotide mutations of the K‐RAS gene (KRAS2) in 2% of pancreatic tumors sampled, but it remained unknown whether these were functional mutations that convert the proto‐oncogene to an oncogene, or unselected mutations that might inactivate protein function. In the current study, the functionality of these rare mutations was quantitated via a mitogen‐activated protein kinase (MAPK) pathway‐specific transactivational reporter system. Pathway activation by dinucleotide mutant proteins was comparable to that of the common G12V mutant K‐Ras protein. Current allele‐specific technologies often employed to detect K‐RAS mutations in clinical tumor samples produce false results when dinucleotide mutations are present. Therefore, it is advisable to consider dinucleotide KRAS2 mutants in the strategic design of mutational screens used to assay clinical tumor samples. Hum Mutat 18:357, 2001. © 2001 Wiley‐Liss, Inc.     

胃黏膜相关淋巴组织型淋巴瘤细胞凋亡和bcl—2、p53蛋白表达

目的 :探讨胃黏膜相关淋巴组织型 (MALT)淋巴瘤中细胞凋亡特点及其与bcl 2、p5 3基因蛋白表达的关系。 方法 :应用TdT酶介导生物素化dUTP缺口末端标记 (TUNEL)技术 ,显示肿瘤凋亡细胞 ,免疫组织化学S P法显示bcl 2、p5 3基因蛋白表达。结果 :低度恶性 ,低高混合恶性 ,以及高度恶性组肿瘤细胞凋亡率平均分别为 (0 2 5± 0 12 ) %、(0 46± 0 2 4) %及 (1 32± 0 35 ) % ;而三组bcl 2阳性率分别为 83%、61 6 %及 43 7%。高度恶性组与低度恶性组bcl 2阳性率及凋亡发生率均差异有显著性 (P <0 0 5 ) ;bcl 2表达与凋亡率呈显著负相关 (P <0 0 5 ) ;86例肿瘤组织中 ,p5 3阳性者 2 7例 (30 % ) ,其中低度恶性组 3例 ,混合恶性组 3例 ,高度恶性组 2 1例。高度恶性组 p5 3阳性率高于其余两组。p5 3表达与bcl 2表达著负相关 (P <0 0 5 )。结论 :在胃MALT淋巴瘤中 ,随着组织学分级的提高 ,凋亡细胞显著增多 ,凋亡在肿瘤发生发展转化中起重要作用。p5 3和bcl 2均为重要的凋亡调控基因 ,在胃MALT淋巴瘤从低度恶性到高度恶性的转化中 ,p5 3和bcl 2基因可能起重要作用。     

Establishment of a human hepatic adenosquamous carcinoma cell line (KMC-2) and its response to cytokines

A human hepatic adenosquamous carcinoma cell line (KMC-2) was established from a serially transplanted tumor in nude mice (nuKMC-2), which originated from human cholangio-cellular carcinoma and showed histological alteration from adenocarcinoma to squamous cell carcinoma (SCC) along with serial transplantation. KMC-2 cells in monolayer culture proliferated in a sheet-like arrangement with a population doubling time of 29.5 h, whereas the cells in 0.1% collagen gel embedded culture formed a compact and tubular structure with the population doubling time of 35.4 h. The cells secreted carbohydrate antigen 19–9 (CA19–9), tissue polypeptide antigen and SCC-related antigen. The back-transplanted nude mouse tumor exhibited morphologic features of adenosquamous carcinoma resembling those in the original nude mouse tumor. IFN-α, IFN-γ and TNF-α suppressed cell proliferation significantly. Functionally, IFN-γ significantly suppressed CA19–9 secretion, and conversely promoted SCC-related antigen secretion. These findings suggest that KMC-2 is the first human hepatic adenosquamous carcinoma cell line primarily originated from adenocarcinoma; the environmental factors, such as the presence of extracellular matrix and the cytokines influenced the growth, morphology and function of KMC-2.