The role of microRNAs in endometrial cancer and influence on future therapy: focusing on miRNA-21.

MicroRNAs are small noncoding polynucleotides, which are involved in numerous biological processes including cell proliferation, differentiation, embryonic development, as well as regulation of cell death and survival. Recent investigations have shown impact of microRNAs on cancers prognosis and diagnosis. Current review focused on the role of microRNA-21 in cancers tumorigenesis. Endometrial cancer is the most common gynecological malignancy and the fourth most common in general classification of cancers in Western Europe; thus discovering new molecules may become a useful diagnostic tool. Furthermore, in this review, the authors emphasized microRNAs having considerable influence on endometrial cancer development. Finally, they highlighted the role of microRNAs as a target for future therapy and circulating microRNAs as a potential biomarker in malignancies.

Molecular and clinical consequences of novel mutations in the arylsulfatase A gene.

Metachromatic leukodystrophy (MLD), a severe neurodegenerative metabolic disorder, is caused by deficient activity of arylsulfatase A (ARSA; EC 3.1.6.8), which leads to a progressive demyelinating process in central and peripheral nervous systems. In this study, a DNA sequence analysis was performed on six Polish patients with different types of MLD. Six novel mutations were identified: one nonsense (p.R114X), three missense (p.G122C, p.G293C, p.C493F) and two frameshift mutations (g.445_446dupG and g.2590_2591dupC). Substitutions p.G293C and p.C493F and duplication g.445_446dupG caused a severe reduction of enzyme activity in transient transfection experiments on mammalian cells (less than 1% of wild-type (WT) ARSA activity). Duplication 2590_2591dupC preserved low-residual ARSA activity (10% of WT ARSA). In summary, the novel MLD-causing mutations in the exons 2, 5 and even in 8 of the ARSA gene described here can be classified as severe type 0, leading in homozygosity to the late infantile form MLD. Growth retardation, delayed motor development, gait disturbances, tonic-clonic seizures and non-epileptic muscle spasms were the first onset symptoms in patients with late infantile form of MLD. In individual with juvenile type MLD gait disturbances evidenced the onset of the disease, while in a patient with late juvenile MLD, difficulties at school were displayed.

Oncogenic tyrosine kinase NPM/ALK induces activation of the MEK/ERK signaling pathway independently of c-Raf.

The mechanisms of cell transformation mediated by the highly oncogenic, chimeric NPM/ALK tyrosine kinase remain only partially understood. Here we report that cell lines and native tissues derived from the NPM/ALK-expressing T-cell lymphoma (ALK+ TCL) display phosphorylation of the extracellular signal-regulated protein kinase (ERK) 1/2 complex. Transfection of BaF3 cells with NPM/ALK induces phosphorylation of EKR1/2 and of its direct activator mitogen-induced extracellular kinase (MEK) 1/2. Depletion of NPM/ALK by small interfering RNA (siRNA) or its inhibition by WHI-154 abrogates the MEK1/2 and ERK1/2 phosphorylation. The NPM/ALK-induced MEK/ERK activation is independent of c-Raf as evidenced by the lack of MEK1/2 and ERK1/2 phosphorylation upon c-Raf inactivation by two different inhibitors, RI and ZM336372, and by its siRNA-mediated depletion. In contrast, ERK1/2 activation is strictly MEK1/2 dependent as shown by suppression of the ERK1/2 phosphorylation by the MEK1/2 inhibitor U0126. The U0126-mediated inhibition of ERK1/2 activation impaired proliferation and viability of the ALK+ TCL cells and expression of antiapoptotic factor Bcl-xL and cell cycle-promoting CDK4 and phospho-RB. Finally, siRNA-mediated depletion of both ERK1 and ERK2 inhibited cell proliferation, whereas depletion of ERK 1 (but not ERK2) markedly increased cell apoptosis. These findings identify MEK/ERK as a new signaling pathway activated by NPM/ALK and indicate that the pathway represents a novel therapeutic target in the ALK-induced malignancies.

Oncogenic tyrosine kinase NPM/ALK induces activation of the rapamycin-sensitive mTOR signaling pathway.

The mechanisms of cell transformation mediated by the nucleophosmin (NPM)/anaplastic lymphoma kinase (ALK) tyrosine kinase are only partially understood. Here, we report that cell lines and native tissues derived from the NPM/ALK-expressing T-cell lymphoma display persistent activation of mammalian target of rapamycin (mTOR) as determined by phosphorylation of mTOR targets S6rp and 4E-binding protein 1 (4E-BP1). The mTOR activation is serum growth factor-independent but nutrient-dependent. It is also dependent on the expression and enzymatic activity of NPM/ALK as demonstrated by cell transfection with wild-type and functionally deficient NPM/ALK, small interfering RNA (siRNA)-mediated NPM/ALK depletion and kinase activity suppression using the inhibitor WHI-P154. The NPM/ALK-induced mTOR activation is transduced through the mitogen-induced extracellular kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway and, to a much lesser degree, through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway. Accordingly, whereas the low-dose PI3K inhibitor wortmannin and Akt inhibitor III profoundly inhibited Akt phosphorylation, they had a very modest effect on S6rp and 4E-BP1 phosphorylation. In turn, MEK inhibitors U0126 and PD98059 and siRNA-mediated depletion of either ERK1 or ERK2 inhibited S6rp phosphorylation much more effectively. Finally, the mTOR inhibitor rapamycin markedly decreased proliferation and increased the apoptotic rate of ALK+TCL cells. These findings identify mTOR as a novel key target of NPM/ALK and suggest that mTOR inhibitors may prove effective in therapy of ALK-induced malignancies.

Tumorigenic conversion of p53-deficient colon epithelial cells by an activated Ki-ras gene.

Distinct genetic abnormalities (loss-of-function mutations of APC and p53 and oncogenic activation of Ki-ras) are associated with specific stages of the sporadic, most common types of colorectal tumors. However, the inability to maintain primary colon epithelial cells in culture has hindered the analysis of the pathogenetic role of these abnormalities in colorectal tumorigenesis. We have now established primary cultures of epithelial cells from the colon crypts of p53-deficient mice; these cells are nontumorigenic as indicated by their failure to form colonies in soft agar and to grow as tumors in immunodeficient SCID mice and in immunocompetent syngeneic hosts. Upon ectopic expression of an activated Ki-ras gene, p53-deficient colon epithelial cells form colonies in soft agar and highly invasive subcutaneous tumors in both immunodeficient and immunocompetent mice. Ectopic expression of wild-type p53, but not of a DNA-binding-deficient mutant, markedly suppressed the colony-forming ability of the Ki-ras-transformed p53-deficient epithelial cells. Together, these findings establish a functional synergism in colorectal tumorigenesis dependent on the effects of an oncogenic Ki-ras in a p53-deficient background. This model of tumorigenic conversion of colon epithelial cells might be useful to identify genetic changes associated with disease progression and to evaluate the therapeutic response to conventional and novel anticancer drugs.

Tumor induced by Moloney sarcoma virus causes periosteal osteogenesis engaging osteopontin, fibronectin, stromelysin-1 and tenascin.

Excessive bone formation occurring in such conditions as paravertebral ligamentous ossification, hallux osteophytes or some neoplastic tumors, presents a significant problem, both epidemiological and clinical. Since pathogenesis of this disorder is still unclear, we studied its mechanism in experimental model utilizing inducible orthotopic osteogenesis. Periosteal bone apposition stimulated by Moloney sarcoma is characterized by unusually high volume of new bone tissue appearing subperiosteally in the bone adjacent to the tumor. Genes engaged in this growth have not been characterized so far. Here we show the results of mRNA Representation Difference Analysis in Moloney sarcoma, which reveal high expression of four genes coding extracellular matrix proteins: osteopontin, fibronectin, stromelysin-1 and tenascin. These findings suggest that the uncommon dynamics of the Moloney sarcoma-induced osteogenesis depends on high expression of these extracellular matrix proteins.

Treatment of Philadelphia leukemia in severe combined immunodeficient mice by combination of cyclophosphamide and bcr/abl antisense oligodeoxynucleotides.

BACKGROUND: Philadelphia cells are human chronic myelogenous leukemia (CML) cells that contain the BCR/ABL oncogene (a fusion of the BCR and ABL genes). Selective eradication of these cells in vitro can be achieved by combined treatment with antisense phosphorothioate oligodeoxynucleotides ([S]ODNs) specifically targeted to this oncogene (bcr/abl [S]ODNs) and a suboptimal (for use as a single agent) dose of mafosfamide (the in vitro active form of cyclophosphamide). PURPOSE: We evaluated the ability of bcr/abl antisense [S]ODNs, alone or subsequent to treatment with a single injection of cyclophosphamide, to suppress the leukemic process induced in severe combined immunodeficient (SCID) mice by Philadelphia cells (i.e., primary CML-blast crisis [CML-BC] cells). In addition, we studied potential mechanisms that might explain the efficacy of the bcr/abl antisense [S]ODN-mafosfamide combination against Philadelphia cells in vitro. METHODS: The effects of treating leukemic mice with cyclophosphamide (25 mg/kg body weight; 25% of the dose required to eradicate evidence of leukemia in SCID mice) and/or bcr/abl antisense [S]ODNs were assessed by analysis of survival, by examination of bone marrow for the presence of leukemia cells (using a colony formation assay or using coupled reverse transcription and the polymerase chain reaction to screen for bcr/abl messenger RNA), and by examination of a variety of tissues for the presence of infiltrating leukemia cells. The induction of apoptosis (a cell death program) in vitro in primary CML-BC cells following treatment with bcr/abl antisense [S]ODNs plus or minus prior treatment with mafosfamide was monitored by use of a commercial assay. Relative cellular uptake of [S]ODNs by CML-BC cells treated in vitro with or without prior treatment with mafosfamide was determined by use of confocal microscopy and flow cytometry (for fluorescent [S]ODNs) or by use of blotting techniques that employed radioactively labeled probes (for extracted, unlabeled [S]ODNs). Levels of specific proteins in treated and untreated cells were determined by use of western blotting methods. Reported P values are two-sided. RESULTS: The disease process in leukemic mice was retarded substantially by combination treatment with cyclophosphamide and specific bcr/abl antisense [S]ODNs (P < .001, relative to treatment with specific antisense [S]ODNs alone, cyclophosphamide alone, or cyclophosphamide plus nonspecific [i.e., control] antisense [S]ODNs); 50% of the mice treated with cyclophosphamide and specific antisense [S]ODNs appeared to be cured of leukemia. The combination treatment was associated with increased induction of apoptosis. In addition, cellular uptake of bcr/abl antisense [S]ODNs appeared to be increased twofold to sixfold by prior treatment with mafosfamide. This increased uptake of [S]ODNs was associated with enhanced suppression of p210bcr/abl protein levels. CONCLUSIONS AND IMPLICATIONS: Combination therapy with antisense [S]ODNs targeted to specific oncogenes and less toxic doses of anticancer drugs may represent a rational strategy to purpose for the treatment of human leukemias.

Non-adherent bone marrow cells are a rich source of cells forming bone in vivo.

Syngeneic, allogeneic and xenogeneic (rat) freshly isolated bone marow cells + stromal cell cultures maintained in vitro for 10-30 days, as well as non-adherent cells removed from these cultures on 3rd-4th day were injected into the kidney parenchyma of mice, immunosuppressed with hydrocortisone. In syngeneic grafts the immunosuppression was omitted. In all transplant systems bone tissue was formed inside the kidney with 20% to 32% variation. Bone produced by allogeneic and xenogeneic cells is subject to rejection when immunosuppression ceases, as the bone formed is of donor origin. The "floating" cells, regardless of the transplant system, normally discarded during media replacement, turned out to be efficient bone producers. This notion is of practical implication when bone marrow cells are used for bone healing.

Results of the administration of bisphosphonates for the prevention of periosteal bone formation following Moloney sarcoma virus-induced tumors.

The effect of continuous and discontinuous administration of bisphosphonates on the periosteal osteogenesis induced by Moloney sarcoma development was studied in mice by histological examination and bone dry mass evaluation. Both EHDP and Cl2MDP did not inhibit periosteal osteogenesis but EHDP, in contrast to Cl2MDP, inhibited the mineralization of osteoid. This inhibition was completely reversible after the withdrawal of the drug. Within two weeks the remineralization of newly formed bone matrix was completed.

Local hypertrophic/hyperplastic changes of keratinizing squamous epithelium of pinna induced by concanavalin A and other immunomodulators in mice.

Intradermal administration of concanavalin A, a potent T-cell mitogen, into an ear lap resulted in activation of chondrogenesis and stimulation of epidermis proliferation. This proliferation is sometimes invasive in character (pearls and epidermal nests form in the underlying connective tissue) but never turns into true cancerous lesions. This reaction can be delayed, but not prevented, by the prostaglandin inhibitor indomethacin. Stimulation of epidermis proliferation was also caused by administration of other immunomodulators, such as carrageenan type IV, Moloney sarcoma development, and rarely in the course of GvHr, but to much lesser degree than with concanavalin A. It is suggested that the same growth factors, which are mediators of local chondrocyte stimulation, are also mediators of keratinocyte activation.

In vivo exposure to sodium fluoride does not modify the yield of viral tumour-induced periosteal bone nor of heterotopic bone induced by human tumour KB cells in mice.

In mice local or systemic administration of fluoride (5-25 mg NaF/kg body weight) during the proliferative phase of bone formation (up to 20 days) has no effect on the yield of bone formed either by local stimulation of periosteal membrane by Moloney sarcoma virus-induced tumour or on bone induced heterotopically by human KB cells. The lack of stimulatory activity of fluoride on rapidly induced osteogenesis in mice is in agreement with recent reports which show that fluoride is not a potent mitogen for human osteoblasts grown in vitro.

Fluvastatin increases heterotopically induced ossicles in mice.

1. The aim of the present study was to evaluate the influence of fluvastatin (3-hydroxy-3-methylglutarylcoenzyme A reductase inhibitor) on heterotopic ossification (HO) induced by HeLa cells. 2. C57Bl/6 mice were injected with 3 x 10(6) HeLa cells into right thigh muscles. Mice in the experimental group received fluvastatin 1.2 mg/kg per day for 17 consecutive days, while mice in the control group received placebo. Intact mice served as an additional control. Seventeen days post-HeLa cell grafting, blood samples were collected to measure total serum cholesterol (TC), triglycerides (TG), low density lipoprotein cholesterol and alkaline phosphatase (AP). 3. In all animals injected with HeLa cells, the mass of mineral deposited in the induced ossicle was established after hydrolysis of soft tissues surrounding the induced ossicles. In fluvastatin-treated mice, the mass of mineral deposited in heterotopically induced ossicles was significantly increased, when compared to mice receiving placebo. This was followed by a significant decrease of TG concentration; whereas the levels of serum AP were not significantly affected. 4. These results indicate that administration of statins may affect heterotopic ossification. This may also have clinical implication, because patients predisposed to HO and receiving statins during hypocholesterolemic treatment, may be at even greater risk of HO.

Molecular background of polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome in a Polish population: novel AIRE mutations and an estimate of disease prevalence.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is an autosomal-recessive autoimmune disease caused by autoimmune regulator gene mutations. The aim of this study was to examine the mutation profile of Polish APECED patients, determine the carrier rate of the most frequent mutation(s) and estimate disease prevalence. While studying 14 unrelated patients, we identified three novel mutations (c.1A>T, affecting the start codon; [IVS1 + 1G>C; IVS1 + 5delG], a complex mutation affecting splice site; c. 908G>C, p.R303P, a missense mutation in plant homeodomain (PHD) and three previously reported mutations (c.769C>T, p.R257X; c.967_979del13bp, C322fsX372; c.931delT, p.C311fsX376). Eleven patients had mutations on both chromosomes, whereas in three patients only a single alteration with proven or likely pathogenic effect was detected. The most frequent was the p.R257X mutation (71% of chromosomes); its carriage rate was assessed in the background population. Analysis of 2008 samples showed eight heterozygotes, indicating the frequency of 0.40% (1:250) and the disease prevalence - 1:129,000 (95% confidence interval: 1:555,000 to 1:30,000). Comparison with an epidemiological estimate (1:619,000, derived for women) suggested that in Poland, APECED is underdiagnosed. Among the patients, no genotype/phenotype correlations were found, but we noted that women had earlier onset of hypoparathyroidism (p < 0.02) and were younger at diagnosis (p < 0.05) than men.

Does colchicine really induce bone formation in the rodent bone marrow?

Intravenous injection of a single dose of colchicine into inbred strains of BALB/c and CFW/L1 mice and into WAG rats did not effect rapid intramedullar bone formation and resorption, as has been claimed by the research group from Tokyo Medical and Dental University [14-17]. The applied doses of colchicine arrested metaphase during the first 4 hours postadministration and were noxious for hemopoietic tissue (necrosis of bone marrow was evident in 2 and 4 day specimens), but on longitudinal, serial sections of long bones there was no evidence of stimulation of osteogenesis at any point in time (2-26-day specimens). It is postulated that the system of ectopic osteogenesis by colchicine injection is not reproducible in mice and WAG rats, and the apparently osteogenic effect of colchicine, observed by the Ogura group [14-17], was mistakenly described as congenital osteopetrosis.

Species differences in uroepithelium-induced bone development: observations on transplants of human uroepithelium in cortisone-treated mice.

Specimens (2-3 x 3-5 mm) of human urinary tract mucosa from bladder (67 cases), ureter and renal pelvis (35 cases) were implanted intramuscularly into cortisone-treated mice in order to elucidate whether human urothelium has the potency to induce osteogenesis. Although xenogeneic epithelium survived up to 14 days after implantation, in none of the 300 implants performed was a bone or cartilage induction observed. Implantation of urinary bladder mucosa from guinea pig and dog resulted in a heterotopic bone and cartilage formation in the graft's vicinity in 28 of 39 cases. The survival of animal transitional epithelium was similar to that of human. It is concluded that human transitional epithelium does not possess osteoinductive potency. Some clinical data pointing to a link between heterotopic osteogenesis and surgical intervention on urinary tract have been reconsidered and explained by the authors' own hypothesis. In addition, data presented in this paper indicate that one should be very careful when attempting to transmit results performed on dog urinary tract to humans.

Is heterotopic bone formation, occurring near a cut ureter, a sign of osteoinductive potency of human urothelium?

A bone formation in the ileo-lumbalis muscle following urine leakage from the injured ureter is reported. The role of osteoinductive potential of urothelium is discussed and negated.

Human urinary bladder-carcinoma cells are non-osteoinductive.

Small pieces (ca. 2-3 x 3-5 mm) of the urinary-tract mucosa from noninvasive papillary transitional-cell carcinomas of the bladder (ca. urotheliale papillare, n = 33), invasive transitional-cell carcinomas of the bladder (ca. urotheliale papillare infiltrans, n = 6, papillary transitional-cell carcinomas of the bladder with squamous metaplasia (ca. urotheliale papillare cum metaplasia planoepitheliale, n = 4), transitional-cell carcinomas in situ (ca. urotheliale in situ, n = 2), and squamous-cell carcinomas of the bladder (ca. planoepitheliale, n = 2) were grafted intramuscularly into cortisone-immunosuppressed mice to test the ability of transformed transitional epithelium to induce heterotopic osteogenesis. Altogether, 156 implants from 47 cases of urinary bladder carcinoma were performed. Histological examination of implants, excised 10-17 days later, revealed relatively good survival of the grafted epithelium, which had proliferated and, in some cases, formed cysts and islands but failed to induce heterotopic osteogenesis in the surrounding host tissues. In nine implants prepared from four cases (noninvasive papillary transitional-cell carcinoma of the bladder and invasive papillary transitional-cell carcinoma of the bladder, two cases each) a small amount of cartilage and/or bone was found in the stroma of grafted tissue. The rarity of this phenomenon--together with the observation that implants of normal human urinary-tract mucosa have never induced the formation of cartilage/bone, whereas in a similar system, dog or guinea-pig grafts are osteogenic--suggests that the cartilage/bone present in the stroma of implanted cancers is the result of metaplasia of the stroma of the neoplasm and not the product of any osteoinductive potency of human urothelium.

Antisense oligodeoxynucleotide combination therapy of primary chronic myelogenous leukemia blast crisis in SCID mice.

The proliferation of chronic myelogenous leukemia (CML) cells and the transformation of normal hematopoietic cells by BCR-ABL appear to require the expression of a functional MYC protein, suggesting an approach to treatment of Philadelphia leukemias based on simultaneous targeting of BCR-ABL and c-MYC. To test this hypothesis, CML-blast crisis (CML-BC) primary cells were treated in vitro with bcr-abl and c-myc antisense phosphorothioate oligodeoxynucleotides ([S]ODNs), individually or in combination. Compared with antisense ODNs targeting of individual oncogenes, downregulation of both BCR-ABL and c-MYC by specific antisense [S]ODNs resulted in a synergistic antiproliferative effect. Colony formation of normal bone marrow cells was not affected by either treatment. To assess the therapeutic potential of multiple oncogene downregulation, SCID mice injected with CML-BC primary cells were treated systematically with equal doses of bcr-abl or c-myc antisense [S]ODNs or with a combination of both antisense [S]ODNs. Compared with mice treated with individual compounds, the disease process was significantly retarded in the group treated with both [S]ODNs as revealed by flow cytometry, clonogenic assay, and RT-PCR analysis to detect leukemic cells in mouse tissue cell suspensions. These effects correlated with a markedly increased survival of leukemic mice treated with both antisense [S]ODNs. Leukemic cells harvested from antisense [S]ODN-treated mice were sensitive to the effects of antisense [S]ODNs in vitro, suggesting that the treatment can be successfully repeated. These data demonstrate the therapeutic potential of targeting multiple cooperating oncogenes.

Blastic transformation of p53-deficient bone marrow cells by p210bcr/abl tyrosine kinase.

Blastic transformation of chronic myelogenous leukemia (CML) is characterized by the presence of nonrandom, secondary genetic abnormalities in the majority of Philadelphia1 clones, and loss of p53 tumor suppressor gene function is a consistent finding in 25-30% of CML blast crisis patients. To test whether the functional loss of p53 plays a direct role in the transition of chronic phase to blast crisis, bone marrow cells from p53+/+ or p53-/- mice were infected with a retrovirus carrying either the wild-type BCR/ABL or the inactive kinase-deficient mutant, and were assessed for colony-forming ability. Infection of p53-/- marrow cells with wild-type BCR/ABL, but not with the kinase-deficient mutant, enhanced formation of hematopoietic colonies and induced growth factor independence at high frequency, as compared with p53+/+ marrow cells. These effects were suppressed when p53-/- marrow cells were coinfected with BCR/ ABL and wild-type p53. p53-deficient BCR/ABL-infected marrow cells had a proliferative advantage, as reflected by an increase in the fraction of S+G2 phase cells and a decrease in the number of apoptotic cells. Immunophenotyping and morphological analysis revealed that BCR/ABL-positive p53-/- cells were much less differentiated than their BCR/ABL-positive p53+/+ counterparts. Injection of immunodeficient mice with BCR/ABL-positive p53-/- cells produced a transplantable, highly aggressive, poorly differentiated acute myelogenous leukemia. In marked contrast, the disease process in mice injected with BCR/ABL-positive p53+/+ marrow cells was characterized by cell infiltrates with a more differentiated phenotype and was significantly retarded, as indicated by a much longer survival of leukemic mice. Together, these findings directly demonstrate that loss of p53 function plays an important role in blast transformation in CML.