Increased gene expression of the ABCC5 transporter without distinct changes in the expression of PDE5 in human cervical cancer cells during growth.

Carcinoma of the uterine cervix represents the second most frequent female malignancy worldwide, but few biochemical tumour markers have been implemented into clinical practice. Elevated extracellular guanosine 3', 5'-cyclic monophosphate (cGMP) levels have been reported to be a sensitive, early and reliable marker for screening relapse in carcinoma of the uterine cervix. The mechanism behind this observation remains unknown. The possibility exists that the cancer cells develop resistance to the antiproliferative effect of high intracellular cGMP levels. The enhanced cGMP expression may originate from either an increase in cellular export capacity by increased expression of member 5 in subfamily C of ATP-Binding-Cassette transporters (ABCC5), or increased substrate (cGMP) levels for this pump. The latter situation occurs with increased expression of inducible nitric oxide synthase (iNOS) and/or soluble guanylyl cyclase (sGC) and/or reduced expression of member 5 of the cyclic nucleotide phosphodiesterases (PDE5). Four transformed human cell lines derived from carcinomas of the uterine cervix (C-4 I, C-33 A, SiHa and ME-180 cells) and one non-transformed human cell line (WI-38) were included in the study in order to unveil which biokinetic components are involved. The expressions of iNOS, sGC, PDE5 and ABCC5 in the initial and final phase of the exponential growth curve were compared. Assuming that the WI-38 control cells mimic the situation in a normal tissue, iNOS remains un-expressed during proliferation, and the expression of sGC is low but shows a clear increase during exponential growth. PDE5 is highly expressed and increases (≈130%) during growth whereas ABCC5 exhibited low to moderate expression, with a moderate increase (≈40%) during growth. The malignant cells exhibited moderate ABCC5 expression with a distinct increase during exponential growth, whereas PDE5 expression remained virtually unchanged. Dysregulation of the cGMP biokinetics in growing malignant cells may account for the elevation of extracellular cGMP observed in patients with carcinoma of the uterine cervix.

Cancer-associated fibroblasts from human NSCLC survive ablative doses of radiation but their invasive capacity is reduced.

BACKGROUND: Cancer-Associated Fibroblasts (CAFs) are significant components of solid malignancies and play central roles in cancer sustainability, invasion and metastasis. In this study we have investigated the invasive capacity and matrix remodelling properties of human lung CAFs after exposure to ablative doses of ionizing radiation (AIR), equivalent to single fractions delivered by stereotactic ablative radiotherapy (SART) for medically inoperable stage-I/II non-small-cell lung cancers. METHODS: CAFs were isolated from lung tumour specimens from 16 donors. Initially, intrinsic radiosensitivity was evaluated by checking viability and extent of DNA-damage response (DDR) at different radiation doses. The migrative and invasive capacities of CAFs were thereafter determined after a sub-lethal single radiation dose of 18 Gy. To ascertain the mechanisms behind the altered invasive capacity of cells, expression of matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs) were measured in the conditioned media several days post-irradiation, along with expression of cell surface integrins and dynamics of focal contacts by vinculin-staining. RESULTS: Exposing CAFs to 1 × 18 Gy resulted in a potent induction of multiple nuclear DDR foci (> 9/cell) with little resolution after 120 h, induced premature cellular senescence and inhibition of the proliferative, migrative and invasive capacity. AIR promoted MMP-3 and inhibited MMP-1 appearance to some extent, but did not affect expression of other major MMPs. Furthermore, surface expression of integrins α2, ß1 and α5 was consistently enhanced, and a dramatic augmentation and redistribution of focal contacts was observed. CONCLUSIONS: Our data indicate that ablative doses of radiation exert advantageous inhibitory effects on the proliferative, migratory and invasive capacity of lung CAFs. The reduced motility of irradiated CAFs might be a consequence of stabilized focal contacts via integrins.

MCF-7 cell apoptosis and cell cycle arrest: non-genomic effects of progesterone and mifepristone (RU-486).

UNLABELLED: The pharmacology of progestins includes actions initiated by various cellular targets, including classic receptors characterized as nuclear transcription factors (nPR), G-protein-coupled membrane receptors (mPR), enzymes, membrane channels and transporters. The effects initiated by targets other than nPR are termed non-genomic and there is an increasing recognition that these effects also play an important role in the regulation of cell growth. MATERIALS AND METHODS: The nPR-positive breast cancer (MCF-7) and the nPR-negative uterine cervix cancer (C4-I) cell lines were exposed to progesterone (PG) and mifepristone (MF) during a culture period of 96 h. Daily cell count, cell cycle analysis and apoptosis assay were performed. RESULTS: It was possible to separate the nPR initiated effects (growth stimulation) from the non-genomic effects (growth inhibition) in the MCF-7 cells. Below 1 µM PG treatment gave a small, but distinct increase in cell density which was effectively blocked by MF. Such an effect was absent from the nPR-negative C4-I cells. For a range of concentrations between 1 µM and 100 µM, the effect of both PG and MF developed over time and showed concentration dependency. The PG concentrations needed to reduce cell density by 50% (IC(50)) were 12.8 ± 1.1 µM and 6.5 ± 0.2 µM for the MCF-7 and C4-I cells, respectively. MF appeared to be equally or slightly more potent, with respective IC(50) values of 6.9 ± 0.5 µM and 5.3 ± 0.3 µM. The cell density reduction was both a result of cell cycle arrest and apoptosis. The combination of PG and MF had a potentiated effect on cell density reduction, cell cycle arrest and apoptosis. CONCLUSION: The antiproliferative/cytotoxic effect of PG and MF in concentrations between 1 and 100 µM is of a non-genomic nature.

Development of a new method to harvest chondroprogenitor cells from underneath cartilage defects in the knees.

BACKGROUND: Mesenchymal progenitor cells from bone marrow hold great potential as a cell source for cartilage repair. Aspiration from the iliac crest is the most widely used method to harvest bone marrow cells for cartilage repair. The objective of our study was to establish a new method to isolate mesenchymal progenitor cells by direct aspiration of bone marrow from the subchondral spongious bone underneath cartilage defects during microfracture treatment and to confirm the chondrogenic potential of the resulting cell cultures. METHODS: Bone marrow was aspirated arthroscopically from patients treated for isolated cartilage defects. Adherent stromal cells were isolated, expanded in monolayer cultures, and characterized by flow cytometry. Chondrogenic induction of cells was achieved by combination of spheroid cultures in hanging drops and the concomitant use of transforming growth factor-beta (TGFbeta). Articular chondrocytes established in three-dimensional (3D) cultures were used as positive cartilage-forming units, and skin fibroblasts were used as negative controls. Three-dimensional constructs were stained for immunohistochemical and histological examination, and a real-time polymerase chain reaction (PCR) was performed to quantify the expression of aggrecan, collagen types 1 and 2, and Sox9. RESULTS: Mesenchymal stem cell-like progenitor cells (MSCs) displaying chondrogenic differentiation capacity were harvested arthroscopically from underneath cartilage lesions on distal femurs using the one-hole technique. Stem cell-related surface antigens analyzed by flow cytometry confirmed the nature of the isolated adherent cells. MSC spheroids stained positive for glycosaminoglycans and collagen type 2. Realtime PCR showed that MSCs in 3D spheroids significantly increased gene expression of collagen type 2, aggrecan, and Sox 9 and down-regulated expression of collagen type 1 when compared to the mRNA levels measured in MSCs monolayers. CONCLUSIONS: We describe a new technique that may be applied for harvesting bone marrow cells from cartilage defects during arthroscopic intervention of the knee. Cells harvested in this way hold full chondrogenic differentiation potential. Our data imply that MSC storage may be established by using marrow from this approach, bypassing the need for cell aspiration from the iliac crest.

Levonorgestrel, medroxyprogesterone and progesterone cause a concentration-dependent reduction in endometrial cancer (Ishikawa) cell density, and high concentrations of progesterone and mifepristone act in synergy.

Endometrial hyperplasia is a precursor lesion of endometrial carcinoma. Clinical studies of endometrial hyperplasia have shown that levonorgestrel (LNG) is more therapeutically effective than medroxyprogesterone acetate (MPA). The present pharmacological in vitro study was performed to compare progestin effects on human endometrial cancer (Ishikawa) cells. Supraphysiological concentrations of progesterone (PG) and high concentrations of LNG and MPA were employed to determine the order of potency in reducing cell density. The order of potency was LNG>MPA>PG with respective 50% inhibitory concentrations (IC(50)) of 3.9+/-0.4, 30.4+/-3.4 and 45.3+/-2.7 microM. Mifepristone (MF) is a potent antiprogestin, but was unable to antagonize the PG-induced cell density reduction. For MF concentrations from 0.2 to 70 microM alone, a PG-mimetic effect was observed with an IC(50) value of 19.0+/-1.7 muM. When PG and MF were combined, a marked reinforcement of the effect was seen. These observations indicate that extranuclear initiated signaling pathways are involved in the reduction of endometrial cancer cells exposed to high concentrations of PG and MF.

Early effects of high concentrations of progesterone and mifepristone A gene expression study of endometrial cancer cells (Ishikawa).

Patients with endometrial hyperplasia representing preliminary stages of endometrial cancer have shown to respond to therapy in 100% of the cases when treated with levonorgestrel-impregnated intrauterine device. Anti-proliferative effect has also been reported after application of an anti-progestin impregnated intrauterine device which showed to induce endometrial atrophy. The intention of the present study was to obtain more information of novel therapeutic targets for hormonal treatment in endometrial hyperplasia and endometrial cancers. Gene expression of signaling pathways after stimulation of Ishikawa cells with high doses of progesterone (32 microM) or Mifepristone (32 microM) was performed. After using an oligo microarrays representing 24,650 human genes and 37,580 gene transcripts, 6154 genes remained after pre-processing and filtering. This resulted in a total of 993 up-regulated genes with 189 genes for progesterone and 255 genes for Mifepristone. The 550 down-regulated genes were distributed with 256 genes for progesterone, 127 genes for RU 486. The results showed that genes presenting the epidermal growth factor (EGF)/MAP-kinase pathway were significantly over-represented by progesterone treatment, whereas, by Mifepristone treatment genes involved in the p53 pathway were also up-regulated (data not shown). These genes may be interesting as potential new therapeutic targets in endometrial hyperplasia and endometrial cancer, as candidate genes for therapy response or as candidate markers for tumor progression.