Patients with limbal stem cell deficiency (LSCD) often experience pain and photophobia due to recurrent epithelial defects and chronic inflammation of the cornea. Successfully restoring a healthy corneal surface in these patients by transplantation of ex vivo expanded human limbal epithelial cells (LECs) may alleviate these symptoms and significantly improve their quality of life. The clinical outcome of transplantation is known to be influenced by the quality of transplanted cells. Presently, several different protocols for cultivation and transplantation of LECs are in use. However, no consensus on an optimal protocol exists. The aim of this study was to examine the effect of culture medium and carrier on the morphology, staining of selected keratins and global gene expression in ex vivo cultured LECs. Limbal biopsies from cadaveric donors were cultured for three weeks on human amniotic membrane (HAM) or on tissue culture coated plastic (PL) in either a complex medium (COM), containing recombinant growth factors, hormones, cholera toxin and fetal bovine serum, or in medium supplemented only with human serum (HS). The expanded LECs were examined by light microscopy (LM), transmission electron microscopy (TEM), immunohistochemistry (IHC) for keratins K3, K7, K8, K12, K13, K14, K15 and K19, as well as microarray and qRT-PCR analysis. The cultured LECs exhibited similar morphology and keratin staining on LM, TEM and IHC examination, regardless of the culture condition. The epithelium was multilayered, with cuboidal basal cells and flattened superficial cells. Cells were attached to each other by desmosomes. Adhesion complexes were observed between basal cells and the underlying carrier in LECs cultured on HAM, but not in LECs cultured on PL. GeneChip Human Gene 2.0 ST microarray (Affymetrix) analysis revealed that 18,653 transcripts were ≥2 fold up or downregulated (p ≤ 0.05). Cells cultured in the same medium (COM or HS) showed more similarities in gene expression than cells cultured on the same carrier (HAM or PL). When each condition was compared to HAM/COM, no statistical difference was found in the transcription level of the selected genes associated with keratin expression, stemness, proliferation, differentiation, apoptosis, corneal wound healing or autophagy. In conclusion, the results indicate that ex vivo cultures of LECs on HAM and PL, using culture media supplemented with COM or HS, yield tissues with similar morphology and keratin staining. The gene expression appears to be more similar in cells cultured in the same medium (COM or HS) compared to cells cultured on the same carrier (HAM or PL).
- MeSH
- biopsie MeSH
- imunohistochemie MeSH
- keratiny biosyntéza genetika MeSH
- kultivační média MeSH
- kultivované buňky MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé středního věku MeSH
- lidé MeSH
- limbus corneae metabolismus ultrastruktura MeSH
- nemoci rohovky genetika patologie chirurgie MeSH
- regulace genové exprese * MeSH
- RNA genetika MeSH
- rohovkový epitel metabolismus ultrastruktura MeSH
- senioři MeSH
- transmisní elektronová mikroskopie MeSH
- transplantace rohovky * MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
Triple-negative breast cancers (TNBC) comprise a heterogeneous subgroup of tumors with a generally poor prognosis. Subclassification of TNBC based on genomic analyses shows that basal-like TNBCs, specifically the basal A or BL2 subtype, are characterized by the expression of ΔNp63, a transcription factor that has been attributed a variety of roles in the regulation of proliferation, differentiation, and cell survival. To investigate the role(s) of p63 in basal-like breast cancers, we used HCC1806 cells that are classified as basal A/BL2. We show that these cells endogenously express p63, mainly as the ΔNp63α isoform. TP63 gene knockout by CRISPR resulted in viable cells that proliferate more slowly and adhere less tightly, with an increased rate of migration. Analysis of adhesion-related gene expression revealed a complex set of alterations in p63-depleted cells, with both increased and decreased adhesion molecules and adhesion substrates compared to parental cells expressing p63. Examination of the phenotype of these cells indicated that endogenous p63 is required to suppress the expression of luminal markers and maintain the basal epithelial phenotype, with increased levels of both CK8 and CK18 and a reduction in N-cadherin levels in cells lacking p63. On the other hand, the level of CK5 was not decreased and ER was not increased, indicating that p63 loss is insufficient to induce full luminal-type differentiation. Taken together, these data demonstrate that p63 exerts multiple pro-oncogenic effects on cell differentiation, proliferation and adhesion in basal-like breast cancers.
- MeSH
- buněčná adheze MeSH
- buněčná diferenciace MeSH
- CD antigeny biosyntéza genetika MeSH
- CRISPR-Cas systémy MeSH
- epitelové buňky metabolismus patologie MeSH
- fenotyp MeSH
- genový knockout MeSH
- kadheriny biosyntéza genetika MeSH
- karcinom patologie MeSH
- keratiny biosyntéza genetika MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nádorové proteiny fyziologie MeSH
- nádorové supresorové proteiny nedostatek fyziologie MeSH
- proliferace buněk MeSH
- protein - isoformy fyziologie MeSH
- regulace genové exprese u nádorů MeSH
- transkripční faktory nedostatek fyziologie MeSH
- triple-negativní karcinom prsu patologie MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
The aim of this study was to detect a spectrum of cytokeratins (CK) present in the adult human cornea, limbus and perilimbal conjunctiva. Cryosections from seven corneo-scleral discs were fixed, and indirect immunofluorescent staining was performed using antibodies directed against CK1-CK10 and CK13-CK20. The percentage of positive cells was calculated in the epithelium of the cornea, limbus and perilimbal conjunctiva. Quantitative real time RT-PCR (qRT-PCR) was used to detect CK6 and CK18 expression in the corneal and conjunctival epithelium. The most intense staining present throughout the cornea was observed for CK3, CK5 and CK14; CK19 was found at the corneal periphery only. CK4 and CK10/13 revealed mild to moderate positivity mostly in the superficial layers of the cornea. The suprabasal cell layers of all examined areas showed a strong positivity for CK16. A heterogeneous staining pattern with a centrifugal decrease in the signal was observed for CK8 and CK18. CK5/6, CK14 and CK19 were present in the limbus, where a positive signal for CK3 was observed in the suprabasal and superficial cells only. In contrast to the cornea, CK15 appeared in the basal and suprabasal layers of the limbus. The perilimbal conjunctiva showed strong immunostaining for CK10/13, CK14 and CK19. A moderate signal for CK7 was detected in the superficial layers of the conjunctiva. qRT-PCR confirmed CK6 and CK18 expression in the corneal and conjunctival epithelium. The detailed characterization of the corneal, limbal and perilimbal conjunctival epithelium under normal circumstances may be useful for characterizing the changes occurring under pathological conditions.
- MeSH
- dospělí MeSH
- fluorescenční mikroskopie MeSH
- fluorescenční protilátková technika nepřímá MeSH
- glyceraldehyd-3-fosfátdehydrogenasy metabolismus MeSH
- imunohistochemie MeSH
- keratiny biosyntéza genetika metabolismus MeSH
- konjunktiva metabolismus MeSH
- lidé středního věku MeSH
- lidé MeSH
- limbus corneae metabolismus MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- RNA biosyntéza genetika MeSH
- rohovka metabolismus MeSH
- rohovkový epitel metabolismus MeSH
- senioři MeSH
- skléra metabolismus MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Carcinoembryonic antigen (CEA) and cytokeratin 20 (CK20) are established tumour markers and the CEA pre-operative levels in serum have prognostic value. The aim of this study was to verify the usefulness of the estimation of CEA and CK20 gene expressions in tissues of colorectal cancers and their liver metastases. PATIENTS AND METHODS: Two hundred and twenty-two specimens of colorectal cancers, their liver metastases, other liver tumours and control tissues were analysed by reverse transcription combined with real-time PCR. RESULTS: The expressions of CEA and CK20 were significantly higher in tumours than in controls; there were differences between tumour types and no relationship to staging or clinical development was found. CK20 expression was inversely dependent on grading. The CEA expression in tumours did not correlate with the CEA levels in serum, but did correlate with serum tissue-specific polypeptide antigen (TPS). CONCLUSION: The measurement of CEA and CK20 gene expressions in tumours did not supply any new prognostic information, but raised the question of the mechanism releasing CEA into the blood.
- MeSH
- adenokarcinom genetika imunologie patologie sekundární MeSH
- dospělí MeSH
- exprese genu MeSH
- financování organizované MeSH
- karcinoembryonální antigen biosyntéza genetika krev MeSH
- keratin-20 MeSH
- keratiny biosyntéza genetika krev MeSH
- kolorektální nádory genetika imunologie krev MeSH
- lidé středního věku MeSH
- lidé MeSH
- messenger RNA biosyntéza genetika MeSH
- nádorové biomarkery krev MeSH
- nádory jater genetika imunologie sekundární MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- staging nádorů MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- senioři nad 80 let MeSH
- senioři MeSH
Three monospecific monoclonal antibodies (BA16, BA17 and A53-B/A2) recognizing different epitopes of the human keratin 19 were used to determine tissue distribution of this 40 kDa keratin polypeptide. Immunohistochemical methods revealed four different staining patterns among normal human epithelial tissues: firstly, complete negativity of the epidermis, sebaceous glands, hepatocytes and other tissues; secondly, homogeneous positivity as seen for example in the gall bladder and urinary bladder epithelium, endometrium and many other epithelia; thirdly, a mosaic of positive and negative cells among mammary gland luminal cells, prostate epithelia and some other epithelia and fourthly, a more complex heterogeneous pattern found in non-keratinizing squamous epithelia and hair follicles with generally the basal layer being the most strongly or sometimes exclusively stained. The pattern seen in non-keratinizing squamous epithelia varied considerably according to the fixation method and the antibody used as well as among different donors and in different areas of the same organ. The other three staining patterns were on the other hand nearly identical with all three antibodies on both frozen sections and sections of methacarn-fixed paraffin-embedded tissues. Our results provide evidence for differential expression of the human keratin 19 at the single cell level, an observation which could be exploited in the study of epithelial differentiation and pathology.
- MeSH
- epitel metabolismus MeSH
- epitopy analýza MeSH
- histocytochemie MeSH
- imunoenzymatické techniky MeSH
- keratiny biosyntéza imunologie MeSH
- lidé MeSH
- monoklonální protilátky * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
