Keratin 19 Expression Research Articles

Nuclear KRT19 is a transcriptional corepressor promoting histone deacetylation and liver tumorigenesis.

Epigenetic reprogramming and escape from terminal differentiation are poorly understood enabling characteristics of liver cancer. Keratin 19 (KRT19), classically known to form the intermediate filament cytoskeleton, is a marker of stemness and worse prognosis in liver cancer. This study aimed to address the functional roles of KRT19 in liver tumorigenesis and to elucidate the underlying mechanisms. Using multiplexed genome editing of hepatocytes in vivo, we demonstrated that KRT19 promoted liver tumorigenesis in mice. Cell fractionation revealed a previously unrecognized nuclear fraction of KRT19. Tandem affinity purification identified histone deacetylase 1 and REST corepressor 1, components of the corepressor of RE-1 silencing transcription factor (CoREST) complex as KRT19-interacting proteins. KRT19 knockout markedly enhanced histone acetylation levels. Mechanistically, KRT19 promotes CoREST complex formation by enhancing histone deacetylase 1 and REST corepressor 1 interaction, thus increasing the deacetylase activity. ChIP-seq revealed hepatocyte-specific genes, such as hepatocyte nuclear factor 4 alpha ( HNF4A ), as direct targets of KRT19-CoREST. In addition, we identified forkhead box P4 as a direct activator of aberrant KRT19 expression in liver cancer. Furthermore, treatment of primary liver tumors and patient-derived xenografts in mice suggest that KRT19 expression has the potential to predict response to histone deacetylase 1 inhibitors especially in combination with lenvatinib. Our data show that nuclear KRT19 acts as a transcriptional corepressor through promoting the deacetylase activity of the CoREST complex, resulting in dedifferentiation of liver cancer. These findings reveal a previously unrecognized function of KRT19 in directly shaping the epigenetic landscape in cancer.

DNA methylation-associated allelic inactivation regulates Keratin 19 gene expression during pancreatic development and carcinogenesis.

Within the pancreas, Keratin 19 (KRT19) labels the ductal lineage and is a determinant of pancreatic ductal adenocarcinoma (PDAC). To investigate KRT19 expression dynamics, we developed a human pluripotent stem cell (PSC)-based KRT19-mCherry reporter system in different genetic backgrounds to monitor KRT19 expression from its endogenous gene locus. A differentiation protocol to generate mature pancreatic duct-like organoids was applied. While KRT19/mCherry expression became evident at the early endoderm stage, mCherry signal was present in nearly all cells at the pancreatic endoderm (PE) and pancreatic progenitor (PP) stages. Interestingly, despite homogenous KRT19 expression, mCherry positivity dropped to 50% after ductal maturation, indicating a permanent switch from biallelic to monoallelic expression. DNA methylation profiling separated the distinct differentiation intermediates, with site-specific DNA methylation patterns occurring at the KRT19 locus during ductal maturation. Accordingly, the monoallelic switch was partially reverted upon treatment with a DNA-methyltransferase inhibitor. In human PDAC cohorts, high KRT19 levels correlate with low locus methylation and decreased survival. At the same time, activation of oncogenic KRASG12D signalling in our reporter system reversed monoallelic back to biallelic KRT19 expression in pancreatic duct-like organoids. Allelic reactivation was also detected in single-cell transcriptomes of human PDACs, which further revealed a positive correlation between KRT19 and KRAS expression. Accordingly, KRAS mutant PDACs had higher KRT19 mRNA but lower KRT19 gene locus DNA methylation than wildtype counterparts. KRT19 protein was additionally detected in plasma of PDAC patients, with higher concentrations correlating with shorter progression-free survival in gemcitabine/nabPaclitaxel-treated and opposing trends in FOLFIRINOX-treated patients. Apart from being an important pancreatic ductal lineage marker, KRT19 appears tightly controlled via a switch from biallelic to monoallelic expression during ductal lineage entry and is aberrantly expressed after oncogenic KRASG12D expression, indicating a role in PDAC development and malignancy. Soluble KRT19 might serve as a relevant biomarker to stratify treatment. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Prognostic significance of KRT19 in Lung Squamous Cancer.

Backgroud: Keratin 19 (KRT19) is the intermediate filament that constitutes the cytoskeleton and regulates cell-cycle and cell death.Objective: We aimed to assess whether KRT19 was involved in lung cancer development.Methods: The expression of KRT19 in lung cancer was evaluated from mRNA expression on open databse and protein abundance on tumor tissue array.Results: Using open microarray gene expression datasets and differential expression analysis, we found that KRT19 was upregulated in lung cancer compared with normal tissue. Further analysis suggested that KRT19 mRNA expression was correlated with tumor progression and overall survival in lung cancer patients. As KRT19 was overexpressed in adenocarcinoma (AC) and squamous cell carcinoma (SCC), we examined the prognostic value of KRT19 protein abundance by tissue microarray (TMA). The results suggested that protein expression of KRT19 was significantly associated with overall survival of SCC.Conclusions: Giving the prognostic role of KRT19 in lung cancer, KRT19 could be considered as an potential molecular marker in lung cancer, especially in SCC.

Silver Sulfadiazine's Effect on Keratin-19 Expression as Stem Cell Marker in Burn Wound Healing.

BackgroundBurn wounds are one of the causes of cutaneous injury that involve both epidermal and dermal layers of skin. Silver sulfadiazine (SSD) has been widely used to treat burn wounds, however recent studies have found the treatment to have some drawbacks, such as cellular toxicity effects. Cutaneous wound regeneration is known to start from the basal layer of the epidermal epithelial cells, which are enriched with highly proliferative cells. Keratin-19 (K19) is one of the epidermal stem cell biomarkers found in the skin. This study aims to explore the expression of K19 in burn wound tissue and to investigate the effect of SSD on its expression.MethodsWe created a burn wound model in Sprague Dawley rats and randomly divided them into control and SSD groups. Wound closure was evaluated (visitrak) overtime series followed by histological evaluation of K19 expression in the wound tissue (immunohistochemistry staining).ResultsOur model successfully represents full-thickness damage caused by a burn wound. The SSD group showed a faster reduction of wound surface area (wound closure) compared to the control group with the peak at day 18 post wounding (p < 0.05). K19 expression was found in both groups and was distributed on epidermal layers, hair follicles and dermis of granulation tissue showing similar patterns.ConclusionTopical application of SSD on burn wounds showed superiority in wound closure and is likely to have no harmful effect on epidermal stem cells. However, further study is required to investigate the effect of silver species on cell viability and toxicity effects during long term treatment.

BRAFV600E-induced KRT19 expression in thyroid cancer promotes lymph node metastasis via EMT.

Keratin 19 (KRT19) is a type I cytokeratin that serves an important role in multiple types of cancer; however, little is known regarding its role in thyroid cancer. Therefore, the aim of the current study was to investigate the role of KRT19 in thyroid cancer. Using The Cancer Genome Atlas database, the expression and clinical significance of KRT19 in thyroid cancer tissues were investigated. The effect of KRT19 in thyroid cancer cell lines was also investigated in vitro. The results demonstrated that KRT19 expression was higher in thyroid cancer when compared with normal thyroid tissues, and was associated with lymph node metastasis, tumor stage and tumor-node-metastasis stage. Knockdown of KRT19 inhibited the proliferation and migration of thyroid cancer cell lines. In addition, increased KRT19 expression was observed only in tumors harboring the B-Raf Proto-Oncogene, Serine/Threonine Kinase (BRAF)V600E mutation, and BRAFV600E overexpression induced KRT19 expression. Furthermore, KRT19 exerted its phenotype via promoting epithelial-mesenchymal transition (EMT). In conclusion, the results of the current study suggest that BRAFV600E-induced KRT19 expression may promote thyroid cancer metastasis via EMT.

Opposing Regulation of Cancer Properties via KRT19-Mediated Differential Modulation of Wnt/β-Catenin/Notch Signaling in Breast and Colon Cancers.

Although Keratin 19 (KRT19) has been reported as a tumor cell marker and found to interact with other proteins that modulate cancer properties, its role in cancer prognosis remains to be fully elucidated. We found that KRT19 expression was increased in both colon and breast cancer, but that knockdown of KRT19 showed opposing effects on cancer properties. In colon cancer, KRT19 knockdown resulted in suppression of cancer via downregulation of Wnt/Notch signaling without altering NUMB transcription. In breast cancer, KRT19 knockdown led to an increase in cancer properties because of attenuated Wnt and enhanced Notch signaling. In colon cancer, KRT19 interacted with β-catenin but not with RAC1, allowing the LEF/TCF transcription factor to bind primarily to the LEF1 and TCF7 promoter regions, whereas in breast cancer, KRT19 interacted with the β-catenin/RAC1 complex and led to apparent upregulation of NUMB expression and NUMB-mediated suppression of Notch signaling. These results reveal a novel differential role of KRT19 in carcinogenesis, due to differential modulation of Wnt/β-catenin/Notch signaling crosstalk through various interactions of KRT19 with only β-catenin or with the β-catenin/RAC1 complex, which might have implications for clinical cancer research.

Dynamic Hydrostatic Pressure Regulates Nucleus Pulposus Phenotypic Expression and Metabolism in a Cell Density-Dependent Manner.

Dynamic hydrostatic pressure (HP) loading can modulate nucleus pulposus (NP) cell metabolism, extracellular matrix (ECM) composition, and induce transformation of notochordal NP cells into mature phenotype. However, the effects of varying cell density and dynamic HP magnitude on NP phenotype and metabolism are unknown. This study examined the effects of physiological magnitudes of HP loading applied to bovine NP cells encapsulated within three-dimensional (3D) alginate beads. Study 1: seeding density (1 M/mL versus 4 M/mL) was evaluated in unloaded and loaded (0.1 MPa, 0.1 Hz) conditions. Study 2: loading magnitude (0, 0.1, and 0.6 MPa) applied at 0.1 Hz to 1 M/mL for 7 days was evaluated. Study 1: 4 M/mL cell density had significantly lower adenosine triphosphate (ATP), glycosaminoglycan (GAG) and collagen content, and increased lactate dehydrogenase (LDH). HP loading significantly increased ATP levels, and expression of aggrecan, collagen I, keratin-19, and N-cadherin in HP loaded versus unloaded groups. Study 2: aggrecan expression increased in a dose dependent manner with HP magnitude, whereas N-cadherin and keratin-19 expression were greatest in low HP loading compared to unloaded. Overall, the findings of the current study indicate that cell seeding density within a 3D construct is a critical variable influencing the mechanobiological response of NP cells to HP loading. NP mechanobiology and phenotypic expression was also found to be dependent on the magnitude of HP loading. These findings suggest that HP loading and culture conditions of NP cells may require complex optimization for engineering an NP replacement tissue.

The Expression of Keratin 19 in Intervertebral Disc Degeneration and Aging

Introduction Low back pain (LBP) affects 80% of the whole population around the world and causes great social-economic burden. Intervertebral disc (IVD) degenerates with aging and is a major contribution to LBP. Mature NP (nucleus pulposus, the center part of IVD) cells are similar to, but not the same as, chondrocytes. The search for NP markers has prompted the proposal of keratin-19 as one of the NP markers mainly based on its transcriptional level.1 In our previous study, among all the investigated. keratin-19 is the most downregulated gene in degenerated human NP. 2 In this study, we aim to track the changes in the protein expression of keratin-19 in disc degeneration and aging process with human clinical specimens and rodent models of induced degeneration and natural aging. Material and Methods The corresponding informed patient consent and institutional review board (IRB) approval and animal ethics approval was obtained from the relevant committee. Human IVD were collected from patients with disc degeneration (graded IV-V at the Schneiderman scale) undergoing discectomy or during corrective scoliosis surgery. Rat induced degeneration model was established by needle puncture as described previously.3 Mice ranged from date of birth to 2 years old were used to represent natural aging in rodents. The expression of keratin-19 was investigated by immunochemical staining. Keratin-19 positivity was determined as the number of keratin-19 positive cells divided by the total number of cells per whole section. Results Keratin-19 expression was detected in both NP and AF (annulus fibrosus) of degenerated and non-degenerated human IVD. The signal of Keratin-19 was exclusively intracellular. Cells within a cluster showed heterogeneous expression for Keratin -19. Degenerated human NP contains significantly less Keratin-19 positive cells when compared with non-degenerated human NP (D-NP versus ND-NP: 13.87 ± 1.27% versus 29.99 ± 4.68%, p = 0.016). In healthy rat, Keratin-19 was found to be strongly expressed in the NP but not in the AF. A minor portion of cells in the cartilaginous EP (endplate) was also Keratin-19 positive. Keratin-19 positivity remained at a similar level post injury in the NP, but were induced in the AF after puncture. In aging mice, at P0, Keratin-19 showed strong expression in the vacuolated notochordal NP cells and in the cartilage anlage of vertebrae bodies, with no expression in the AF. From P0 to 18 months of age, Keratin-19 remained expressed at a relatively constant level in the NP constituted by large vacuolated notochordal cells. By 24 months of age, Keratin-19 expression decreased in the NP. Conclusion We found that although Keratin-19 was significantly decreased in human degenerated NP, it only showed a mild decrease or otherwise unchanged expression in rodent models of progressive disc degeneration and aging. Since Keratin-19 is also a notochordal cell marker,4 it may reflect that notochordal cells are still present in the aged mouse NP and during the mild stage of degeneration in the rat model. To our knowledge, this is the first investigation into the expression and location of keratin-19 in different IVD aging/degeneration systems. Acknowledgments This work was supported by General Funding from National Science Foundation of China (NSFC, 81371993), Small Project Funding of The University of Hong Kong (201409176191), and General Research Fund of RGC in Hong Kong (17104815). References Lv F, Leung VY, Huang S, Huang Y, Sun Y, Cheung KM. In search of nucleus pulposus-specific molecular markers. Rheumatology (Oxford) 2014;53(4):600–610 Lv F, et al. Global Spine Congress, Hong Kong; 2013 Leung VY, et al. Stem Cells 2014;218(1):113 Weiler C, Nerlich AG, Schaaf R, Bachmeier BE, Wuertz K, Boos N. Immunohistochemical identification of notochordal markers in cells in the aging human lumbar intervertebral disc. Eur Spine J 2010;19(10):1761–1770

Abstract 538: Identification and validation of novel prostate cancer biomarkers using the Berg Interrogative Biology™ platform

Abstract Prostate cancer is the most frequent cancer diagnosis among men and the second leading cause of cancer-related death. Despite the widespread use of digital rectal exam (DRE) and blood-based screening of prostate-specific antigen (PSA) for prostate cancer screening, there are significant limitations in their specificity and prognostic value. Biomarkers which distinguish i) PSA-low prostate cancer from benign prostatic hyperplasia (BPH), and ii) indolent versus aggressive disease course represent unmet clinical needs. Experimentally, a panel of prostate cancer cell lines and non-tumorigenic, human primary cells were exposed to in vitro conditions designed to simulate poor oxygenation, low pH, diminished nutrient microenvironments, and metabolic perturbations (24-48 h) followed by iTRAQ proteomic analysis of cell lysates. Using the Berg Interrogative Biology™ platform, proteomic data were then subjected to Bayesian network learning to map molecular interactions, with cytoskeletal and scaffolding proteins Filamin A (FLNA), Filamin B (FLNB), and Keratin 19 (KRT19) identified as candidate prostate cancer biomarkers. To validate biomarker expression, mRNA and protein was quantified in panel of primary human prostate epithelial cells (HPrEC) and androgen-sensitive (LnCAP) or refractory (DU145, PC-3) prostate cancer cells, and each was differentially detected in one or more prostate cancer cell lines compared to HPrEC. Using proteomic analysis, peptides from FLNA, FLNB, and KRT19 were also detected cell culture media conditioned by prostate cancer cells (24 h), indicating they can be secreted. Importantly, unlike PSA expression, global regulation of FLNA, FLNB, and KRT19 expression remained unaltered after treatment with multiple prostate-cancer relevant stimuli (e.g., hypoxia, androgens, and inflammatory stimuli). In vivo validation was next conducted in sera from men (N = 447) with confirmed prostate cancer, benign prostate tumors, or BPH using LDT ELISA assays in a CLIA-certified laboratory. To assess the sensitivity and specificity of FLNA, FLNB, and KRT19 compared to PSA, ROC curve analysis was performed. The individual predictive power of each biomarker alone was comparable to that of PSA. However, the combination of age, levels of FLNA, FLNB, and KRT19, and PSA out-performed PSA alone in identification of patients with prostate cancer stratified compared to benign status, gleason scores and incidence of BPH. Together, these data validate the use of the Berg Interrogative Biology™ platform for biomarker discovery and indicate that FLNA, FLNB, and KRT19 can be used in conjunction with PSA for more sensitive and specific prostate cancer screening, a critical unmet need in the field. Citation Format: Niven R. Narain, Anne Diers, Rakibou Ouro-Djobo, Joyce Chan, Leonardo O. Rodrigues, Vivek K. Vishnudas, Eleftherios P. Diamandis, Viatcheslav R. Akmaev, Rangaprasad Sarangarajan. Identification and validation of novel prostate cancer biomarkers using the Berg Interrogative Biology™ platform. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 538. doi:10.1158/1538-7445.AM2015-538

Evaluation of CGB, GNRH1, MET and KRT19 Genes Expression Profile as a Circulating Tumor Cells Marker in Blood of Cancer Patients

The aim of this study was to evaluate human chorionic gonadotropin beta subunit (CGB), gonadotropin releasing hormone type 1 (GNRH1), hepatocyte growth factor receptor (MET) and keratin 19 (KRT19) genes expression profile as a circulating tumor cells (CTC) marker in blood of cancer patients. Expression level of studied genes was assessed in peripheral blood of 122 patients with different types of cancers and 86 healthy volunteers using reverse transcription real time quantitative polymerase chain reaction (RT-qPCR). The result of the experiments demonstrated that in blood of cancer patients the level of MET transcripts showed positive correlation with KRT19 and negative correlation with GNRH1. In the control group negative correlation between CGB and GNRH1 was documented. What is more the level of CGB, MET and KRT19 expression was significantly higher in blood of cancer patients. Even though the analysis proved that studied genes are expressed in blood of both cancer patients and healthy volunteers, their expression level was highly heterogeneous. In order to interpret the results, the obtained data was log-transformed and fitted to multiplied normal distribution model using the maximal likelihood method. The results of this analysis showed elevated expression of CGB, MET and KRT19 together with extremely high levels of GNRH1 in blood of cancer patients which might indicate the presence of circulating tumor cells and increased risk of metastasis.

Keratin 19 expression correlates with poor prognosis in breast cancer.

Breast cancer expression profiling has been used for determining biomarkers. Using gene expression profiles of 2,400 patients we identified keratin 19 (KRT19) as a highly deregulated gene in breast cancer. KRT19 expression is independent of patient race but correlates with disease grade, and ER, PR or HER2 expression. Expression of TPD52, GATA3 and KRT18 was increased in KRT19 expressing patients. Furthermore, KRT19 expression was associated with ER up-regulation and Luminal B gene signatures, as well as a constitutive RAF1 signaling pathway. Finally, KRT19 expression correlated with poor overall survival. Taken together, our results suggest that KRT19 expression can be used as a prognostic marker.

Squamous Cell/Adenosquamous Carcinomas and Adenocarcinomas of the Gallbladder: An Immunohistochemistry Study of Prognostic Markers

Gallbladder cancers (GBCs) are highly aggressive and lethal diseases. However, the key molecular mechanisms responsible for the progression and prognosis of GBCs have not been identified. No biological markers for effectively identifying GBC subtypes have been reported. In this study the expression of keratin 19 (KRT19) and human achaete-scute homolog 1 (hASH1) proteins in 46 squamous cell/adenosquamous carcinomas (SC/ASC) and 80 adenocarcinomas (AC) were examined using immunohistochemistry. Negative KRT19 or positive hASH1 expression were significantly associated with lymph node metastasis, invasion and TNM stage of SC/ASC patients. In contrast, positive KRT19 and hASH1 expression were significantly associated with large tumor size, lymph metastasis, invasion, and TNM stage in AC patients. Univariate Kaplan-Meier analysis showed that loss of KRT19 or elevated hASH1 expression significantly correlated with decreased survival in SC/ASC patients. In contrast, positive KRT19 and hASH1 expression correlated with a shorter survival time in AC patients. Multivariate Cox regression analysis showed that negative KRT19 expression or positive hASH1 expression was an independent poor-prognostic predictor in SC/ASC, but positive KRT19 and hASH1 expression were poor-prognostic factors in AC patients. Our study suggested that hASH1 can be used to determine the malignancy of SC/ASC and AC tumors and is associated with poor prognosis. In contrast, KRT19 is a protective factor in AC patients but a sign of malignancy in SC/ASC patients.

Expression of caspase-14 and keratin-19 in the human epidermis and appendages during fetal skin development

Caspase-14 is a seemingly non-apoptotic caspase involved in keratinocyte differentiation and cornification of the skin. Keratin-19 is an epithelial marker and a potential marker of epidermal stem cells that is expressed during human fetal skin development. We examined the immunohistochemical expression of caspase-14 in relation to CK-19 in the human fetal skin during development and perinatally, to assess their role in human skin maturation. Skin samples were received at autopsy. In the fetal epidermis, caspase-14 was predominantly expressed in the more differentiated layers, gradually disappearing from the basal layer toward term. By contrast, keratin-19 expression gradually decreased with epidermal maturation through gestation (rho = -0.949; p = 0.0001) and was a marker of the germinative layers. Keratin-19 was preserved in scarce basal cell nests at term and postnatally. Caspase-14 and keratin-19 were inversely expressed in the differentiating epidermal layers through gestation (p < 0.0001). Concerning the appendages, in hair follicles and sebaceous glands, caspase-14 located preferentially in the more differentiated layers of the inner root sheath, whereas keratin-19 was expressed in the outer sheath. Eccrine sweat glands showed a variable pattern of caspase-14 and keratin-19 expression. In conclusion, caspase-14 emerged as a marker of human skin differentiation during development, while keratin-19 marked the germinative epithelial layers in the fetal epidermis and appendages and possibly the nests of epidermal stem cells.

Human Galectins Induce Conversion of Dermal Fibroblasts into Myofibroblasts and Production of Extracellular Matrix: Potential Application in Tissue Engineering and Wound Repair

Members of the galectin family of endogenous lectins are potent adhesion/growth-regulatory effectors. Their multifunctionality opens possibilities for their use in bioapplications. We studied whether human galectins induce the conversion of human dermal fibroblasts into myofibroblasts (MFBs) and the production of a bioactive extracellular matrix scaffold is suitable for cell culture. Testing a panel of galectins of all three subgroups, including natural and engineered variants, we detected activity for the proto-type galectin-1 and galectin-7, the chimera-type galectin-3 and the tandem-repeat-type galectin-4. The activity of galectin-1 required the integrity of the carbohydrate recognition domain. It was independent of the presence of TGF-β1, but it yielded an additive effect. The resulting MFBs, relevant, for example, for tumor progression, generated a matrix scaffold rich in fibronectin and galectin-1 that supported keratinocyte culture without feeder cells. Of note, keratinocytes cultured on this substratum presented a stem-like cell phenotype with small size and keratin-19 expression. In vivo in rats, galectin-1 had a positive effect on skin wound closure 21 days after surgery. In conclusion, we describe the differential potential of certain human galectins to induce the conversion of dermal fibroblasts into MFBs and the generation of a bioactive cell culture substratum.

Expression of Heterochromatin Protein 1 in the Primary Tumor of Breast Cancer Patients in the Presence or Absence of Occult Metastatic Cells in the Bone Marrow

Gene silencing may occur in breast cancer samples from patients presenting with occult metastatic cells in the bone marrow and one mechanism regulating gene suppression is heterochromatin formation. We have studied whether members of the heterochromatin protein 1 family (HP1Hs alpha, HP1Hs beta and HP1Hs gamma), which take part in chromatin packaging and gene expression regulation, were differentially expressed in tumors from patients with and without occult metastatic cells in their bone marrow. Tumor samples and bone marrow aspirates were obtained from 37 breast cancer patients. Median age was 63 years and 68% of the patients presented with clinical stage I/II disease. Presence of occult metastatic cells in bone marrow was detected through keratin-19 expression by nested RT-PCR in samples from 20 patients (54.1%). The presence of occult metastatic cells in bone marrow was not associated with node involvement, histological grade, estrogen receptor and ERBB2 immunoexpression. Relative gene expression of HP1Hs alpha, HP1Hs beta and HP1Hs gamma was determined by realtime RT-PCR and did not vary according to the presence of occult metastatic cells in bone marrow. In addition, the combined expression of these three transcripts could not be used to classify samples according to the presence of bone marrow micrometastasis. Our work indicates that regulation of heterochromatin formation through HP1 family members may not be the sole mechanism implicated in the metastatic process to the bone marrow.

Expression and significance of cartilage oligomeric matrix protein, C-X-C motif 9 and keratin 19 in oral submucosal fibrosis

To investigate the expression and localization of cartilage oligomeric matrix protein (COMP), C-X-C motif 9 (CXCL9) and keratin 19 (KRT19) in oral submucous fibrosis (OSF) and to evaluate their roles in the pathopoiesis of OSF. The expression and localization of COMP, CXCL9 and KRT19 were investigated in the specimens of 66 patients with oral submucous fibrosis and 14 normal controls by immunohistochemistry, and their protein and mRNA expressions were detected by Western blotting and RT-PCR. COMP was overexpressed in 36 (55%) cases of OSF, and 43 (65%) cases showed positive immunoreactivity for CXCL9 protein in the cytoplasm of inflammatory cells and endothelial cells in OSF. All normal buccal mucosa tissues were stained continuously and strongly for KRT19 in the cytoplasm of basal cells, only 7 (11%) of 66 OSF samples showed faint and fragmented KRT19 staining in the cytoplasm of basal cells. RT-PCR and Western blotting results were fully consistent with the immunohistochemical data. COMP, CXCL9 and KRT19 play an important role in the pathopoiesis of OSF.

The search for cancer stem cells in hepatocellular carcinoma

Recent evidence suggests that some solid cancers originate from cancer stem cells. We have identified a subset of candidate stem cells, which are termed side population (SP) cells, in the hepatocellular carcinoma cell line Huh7. Because most stem cells reside in the G0 phase of the cell cycle, G0 cells were isolated, and the relationship between SP cells and G0 cells was investigated to clarify the biological characteristics of G0 cells. Huh7 cells were sorted using Hoechst 33342 and Pyronin Y. The cells were then divided into G0, G1, and G2/M fractions and cultured under low-attachment conditions to obtain cellular spheres. Tumorigenetic ability was investigated using subcutaneous transplantation to NOD/SCID mice. G0 and G1 cells were analyzed for markers indicative of hepatocytic (albumin expression) and cholangiocytic (keratin 19 expression) differentiation and DNA synthesis (Ki67). The cell-cycle distribution of cultured Huh7 cells was 0.7% (G0), 63.8% (G1), and 34.5% (G2/M, S). The G0 cells were located within the neck of the SP fraction. The G0 cells showed spheroid formation and 3-dimensional growth. Those cells showed marked tumorigenesis in NOD/SCID transplantation. G0 cells, which did not express Ki67, were weakly positive for expression of albumin and were clearly positive for the expression of keratin 19. In contrast, G1 cells were positive for Ki67 and albumin expression but negative for keratin 19. G0 cells are present in the SP fraction of Huh7. They show self-renewal, tumorigenesis, and bidirectional lineage. These findings suggest that the G0 cells within the Huh7 cell line are promising candidates as cancer stem cells for future studies of hepatocellular carcinoma.

109. Search of Cancer Stem Cell in Hepatocellular Carcinoma

Background and Aims: Recent evidence suggests that some solid cancers may originate from cancer stem cells. We have identified subset of candidate stem cells termed side population (SP) cells in Huh7, a hepatocellular carcinoma cell line (Stem Cells 2006). On the other hand, stem cells are considered to exist at G0 phase of cell cycle. In this study, we investigated the relationship between SP cells and G0 cells, and clarified the biological characteristics of G0 phase cells. Study Design: Huh7 cells were sorted by flow cytometry using Hoechst 33342 and pyronin Y. Then, the cells were divided into G0, G1, and G2/M phase, respectively, and also into SP and non-SP fractions. The cells were cultured under a low attached condition to obtain spheroid cell clusters, and SP and G0 cells were counted. The cells in each cell-cycle phase were extracted respectively, and analyzed with respect to DNA synthesis condition (Ki67), hepatocytic (albumin expression) or cholangiocytic (keratin19 expression) differentiations. Results: The proportion of each cell-cycle phase of the Huh7 cells was 0.7%, 63.8%, and 34.5%, in G0, G1, and G2/M, respectively. On the other hand, SP and non-SP fraction were recognized in 0.9% and 98.8%, respectively. Interestingly, the G0 phase cells were located at a neck of SP fraction. Under a condition of spheroid cell culture, the ratio of SP fraction and that of G0 fraction were significantly increased. The G0 cells showed negative Ki67 expression, weakly positive albumin expression, and positive keratin19 expression, whereas, the G1 and G2/M cells did positive Ki67 expression, albumin expression, but negative keratin19 expression. Conclusion: It is demonstrated that Huh7 cells in G0 phase were included in the SP fraction and that they showed less proliferation activity. These cells, however, showed bidirectional differentiation to hepatocyte and cholangiocyte lineages. The findings suggest the G0 cells of Huh7 are promising candidate of cancer stem cells in hepatocellular carcinoma.

Expression of heterochromatin protein 1 in the primary tumor of breast cancer patients in the presence or absence of occult metastatic cells in the bone marrow

Gene silencing may occur in breast cancer samples from patients presenting with occult metastatic cells in the bone marrow and one mechanism regulating gene suppression is heterochromatin formation. We have studied whether members of the heterochromatin protein 1 family (HP1Hs alpha, HP1Hs beta and HP1Hs gamma), which take part in chromatin packaging and gene expression regulation, were differentially expressed in tumors from patients with and without occult metastatic cells in their bone marrow. Tumor samples and bone marrow aspirates were obtained from 37 breast cancer patients. Median age was 63 years and 68% of the patients presented with clinical stage I/II disease. Presence of occult metastatic cells in bone marrow was detected through keratin-19 expression by nested RT-PCR in samples from 20 patients (54.1%). The presence of occult metastatic cells in bone marrow was not associated with node involvement, histological grade, estrogen receptor and ERBB2 immunoexpression. Relative gene expression of HP1Hs alpha, HP1Hs beta and HP1Hs gamma was determined by realtime RT-PCR and did not vary according to the presence of occult metastatic cells in bone marrow. In addition, the combined expression of these three transcripts could not be used to classify samples according to the presence of bone marrow micrometastasis. Our work indicates that regulation of heterochromatin formation through HP1 family members may not be the sole mechanism implicated in the metastatic process to the bone marrow.