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1.
Cell Prolif ; 41(3): 554-73, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18422703

ABSTRACT

OBJECTIVES: Polycystin-1 (PC1), a signalling receptor regulating Ca(2+)-permeable cation channels, is mutated in autosomal dominant polycystic kidney disease, which is typically characterized by increased cell proliferation. However, the precise mechanisms by which PC1 functions on Ca(2+) homeostasis, signalling and cell proliferation remain unclear. Here, we investigated the possible role of PC1 as a modulator of non-capacitative Ca(2+) entry (NCCE) and Ca(2+) oscillations, with downstream effects on cell proliferation. RESULTS AND DISCUSSION: By employing RNA interference, we show that depletion of endogenous PC1 in HEK293 cells leads to an increase in serum-induced Ca(2+) oscillations, triggering nuclear factor of activated T cell activation and leading to cell cycle progression. Consistently, Ca(2+) oscillations and cell proliferation are increased in PC1-mutated kidney cystic cell lines, but both abnormal features are reduced in cells that exogenously express PC1. Notably, blockers of the NCCE pathway, but not of the CCE, blunt abnormal oscillation and cell proliferation. Our study therefore provides the first demonstration that PC1 modulates Ca(2+) oscillations and a molecular mechanism to explain the association between abnormal Ca(2+) homeostasis and cell proliferation in autosomal dominant polycystic kidney disease.


Subject(s)
Calcium Signaling , Kidney/pathology , TRPP Cation Channels/metabolism , Cell Line , Cell Line, Transformed , Cell Proliferation , Codon, Nonsense/genetics , Cytoplasm/metabolism , Enzyme Activation , Humans , Kidney/enzymology , Models, Biological , NFATC Transcription Factors/metabolism , Polycystic Kidney, Autosomal Dominant/enzymology , Polycystic Kidney, Autosomal Dominant/pathology , Protein Kinase C-alpha/metabolism , RNA Interference
2.
J Dent Res ; 85(4): 354-8, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16567558

ABSTRACT

Biostite is a hydroxyapatite-derived biomaterial that is used in periodontal and bone reconstructive procedures due to its osteoconductive properties. Since the molecular effects of this biomaterial on osteoblasts are still unknown, we decided to assess whether it may specifically modulate osteoblast functions in vitro. We found that a brief exposure to Biostite significantly reduced the proliferation of MG-63 and SaOS-2 osteoblast-like cells to approximately 50% of the plateau value. Furthermore, gene array analysis of MG-63 cells showed that Biostite caused a differential expression of 37 genes which are involved in cell proliferation and interaction, and related to osteoblast differentiation and tissue regeneration. Results were confirmed by RT-PCR, Western blot, and by an increase in alkaline phosphatase (ALP) specific activity. Biostite also increased levels of polycystin-2, a mechano-sensitive Ca(2+) channel, a promising new marker of bone cell differentiation. Biostite, therefore, may directly affect osteoblasts by enhancing chondro/osteogenic gene expression and cytoskeleton-related signaling pathways, which may contribute to its clinical efficacy.


Subject(s)
Bone Substitutes/pharmacology , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Collagen/pharmacology , Glycosaminoglycans/pharmacology , Hydroxyapatites/pharmacology , Osteoblasts/drug effects , Alkaline Phosphatase/drug effects , Chondrogenesis/drug effects , Chondrogenesis/genetics , Cytoskeleton/drug effects , Cytoskeleton/genetics , Gene Expression/drug effects , Gene Expression Profiling , Humans , Osteoblasts/cytology , Osteoblasts/enzymology , Osteogenesis/drug effects , Osteogenesis/genetics , Prostheses and Implants , Tumor Cells, Cultured
3.
Leukemia ; 16(5): 886-93, 2002 May.
Article in English | MEDLINE | ID: mdl-11986951

ABSTRACT

HTIF1alpha, a transcription coactivator which is able to mediate RARalpha activity and functionally interact with PML, is encoded by a gene on chromosome 7q32-34, which is a critical region in acute myeloid leukemias (AML). With the assumption that this gene may be related to AML, we investigated the HTIF1alpha DNA structure and RNA expression in leukemic cells from 36 M1-M5 AML patients (28 "de novo" and eight "secondary" to myelodysplastic syndrome (MDS)). Abnormal HTIF1alpha DNA fragments were never found, whereas loss of HTIF1alpha DNA was observed in the patients with chromosome 7q32 deletion and translocation, and in one case without detectable chromosome 7 abnormality. HTIF1alpha RNA was found in acute myelocytic leukemic blasts, and was almost undetectable in normal mononuclear cells. The expression varied among the patients: higher in M1 to M3 subtypes, with the highest values in M1; low levels were constantly observed in M4 and M5 AML. In addition, HTIF1alpha was significantly overexpressed in MDS-related AML (MDR-AML), but not in MDS. We also found that HTIF1alpha expression was high in myeloid cell lines. In myeloblastic HL60 and promyelocytic NB4 cells, induced to differentiate along the monocytic-macrophage pathway by TPA or vitamin D3, HTIF1alpha expression decreased, whereas it was maintained at high levels on induction to granulocytic differentiation by RA or DMSO. In K562 cells, HTIF1alpha RNA levels did not change after hemin-induced erythroid differentiation. These results suggest that HTIF1alpha could play a role in myeloid differentiation, being distinctly regulated in hematopoietic lineages.


Subject(s)
Gene Expression Regulation , Leukemia, Myeloid/genetics , Myelodysplastic Syndromes/genetics , Nuclear Proteins/genetics , Transcription Factors/genetics , Acute Disease , Adult , Aged , Aged, 80 and over , Blood Cells/pathology , Bone Marrow/pathology , Case-Control Studies , Cell Differentiation , Chromosome Aberrations , Chromosomes, Human, Pair 7/genetics , DNA/chemistry , Female , Humans , Leukemia, Myeloid/classification , Leukemia, Myeloid/etiology , Male , Middle Aged , Nuclear Proteins/physiology , Protein Isoforms/genetics , RNA, Messenger/metabolism , Transcription Factors/physiology , Tumor Cells, Cultured
4.
J Endocrinol ; 172(3): 683-93, 2002 Mar.
Article in English | MEDLINE | ID: mdl-11874716

ABSTRACT

Estrogen receptor (ER) alpha is expressed during osteoblast differentiation; however, both its functional role in bone metabolism and its involvement in osteoporotic pathogenesis caused by estrogen deficiency are not well understood. Loss of ER alpha gene expression could be one of the mechanisms leading to osteoporosis. Therefore, we investigated a possible modulation of ER alpha gene expression in a human osteoblastic cell line and in four primary osteoblast cultures by using a decoy strategy. Double stranded DNA molecules, mimicking a regulatory region of the ER alpha gene promoter (DNA-102) and acting as a 'silencer' in breast cancer cells, were introduced into osteoblasts as 'decoy' cis-elements to bind and functionally inactivate a putative negative transcription factor, and thus to induce ER alpha gene expression. We found that the DNA-102 molecule was able to specifically bind osteoblast nuclear proteins. Before decoy treatment, absence or variable low levels of ER alpha RNAs in the different cultures were detected. When the cells were transfected with the DNA-102 decoy, an increase in expression of ER alpha and osteoblastic markers, such as osteopontin, was observed, indicating a more differentiated osteoblastic phenotype both in the cell line and in primary cultures. These results showed that the DNA-102 sequence competes with endogenous specific negative transcription factors that may be critical for a decrease in or lack of ER alpha gene transcription. Therefore, osteoblastic transfection with the DNA-102 decoy molecule may be considered a tempting model in a putative therapeutic approach for those pathologies, such as osteoporosis, in which the decrease or loss of ER alpha expression plays a critical role in bone function.


Subject(s)
Gene Expression Regulation , Osteoclasts/metabolism , Promoter Regions, Genetic , Receptors, Estrogen/genetics , Biomarkers/analysis , Blotting, Western , Cell Differentiation , Cells, Cultured , Electrophoretic Mobility Shift Assay , Estrogen Receptor alpha , Humans , Models, Biological , Osteoclasts/cytology , Osteonectin/analysis , Osteopontin , Osteoporosis/genetics , Osteoporosis/metabolism , Osteoporosis/therapy , Receptors, Estrogen/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sialoglycoproteins/analysis
5.
Leukemia ; 15(8): 1262-7, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11480569

ABSTRACT

A novel recurrent translocation t(11;14)(p11;q32) was found in three patients with splenic marginal zone B cell lymphoma (MZBCL). Fluorescence in situ hybridization (FISH) studies with IgH probes revealed in all cases involvement of the IgH locus, with breakpoint downstream of the IGVH sequences. Partner genes at 11p11 were not identified. The translocation defined the stem line in two patients, who carried additional cytogenetic aberrations, including a 17p deletion, present in both cases. In one patient a 7q- chromosome was the primary cytogenetic defect, the t(11;14) having been found in four out of 11 abnormal metaphase cells at the time of transformation into high-grade MZBCL. Hematological features in all cases included splenomegaly with peripheral blood (PB) involvement by a monoclonal B cell population consisting of lymphocytes with villous projections and several blast-like cells. The immunophenotype was CD19+; CD22bright+; CD23-, CD10-, CD5-, surface Igbright+. A bone biopsy in one patient revealed an interstitial infiltration with an intrasinusoidal pattern of growth. Histological studies on spleen specimens in two patients showed an expanded marginal zone, with small lymphocytes and several blast-like cells. One patient had a therapy-demanding disease, with partial, short-term responses to cytotoxic treatment; one patient transformed into a high-grade MZBCL involving the gut, the PB and the bone marrow 2 years after diagnosis; one patient was unresponsive to cytotoxic treatment and underwent splenectomy. The t(11;14)(p11;q32) may define a subset of splenic MZBCL with a high-grade component and a relatively aggressive clinical behavior.


Subject(s)
Chromosomes, Human, Pair 11 , Chromosomes, Human, Pair 14 , Lymphoma, B-Cell/genetics , Splenic Neoplasms/genetics , Translocation, Genetic , Aged , Aged, 80 and over , Antigens, CD/immunology , Female , Humans , Immunophenotyping , Lymphoma, B-Cell/immunology , Lymphoma, B-Cell/pathology , Male , Middle Aged , Splenic Neoplasms/immunology , Splenic Neoplasms/pathology
7.
Hum Mutat ; 16(5): 444-5, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11058904

ABSTRACT

Autosomal dominant polycystic kidney disease (ADPKD) is a common disorder mostly characterized by cyst formation in kidney tubules. The majority of ADPKD cases is caused by mutations in the PKD1 gene, but no prevalent mutation has been reported. By heteroduplex analysis of the 3' single-copy region of the gene, we have searched for mutations in subjects from 40 ADPKD families of Northern Italy. Seven novel polymorphisms and three novel disease-associated mutations (R3718Q, L3851P and IVS45+56del25) were identified. Both missense mutations are located in the major extracellular loop of polycystin-1. The 25 bp deletion inside intron 45 did not affect 5' and 3' consensus splicing sites, but caused a 56 nucleotide out of frame-deletion due to activation of a cryptic 3' splice site in exon 46. The mutated RNA should produce a truncated polycystin 1 at the G binding peptide in the intracellular C-terminal end of the protein. RT-PCR analysis showed that the disease-associated mutations were present in transcribed sequences. In particular, RNA analysis of BHK cells transfected with PKD1 genomic DNA, including the deleted intron, showed that no normal transcript is produced by the deleted gene. This intronic mutation, found in a large pedigree, seems to be associated with a prevalence of cerebrovascular disease.


Subject(s)
Alternative Splicing/genetics , Gene Expression/genetics , Mutation, Missense/genetics , Polycystic Kidney, Autosomal Dominant/genetics , Protein Biosynthesis , Proteins/genetics , Adult , Aged , Base Sequence/genetics , Female , Humans , Kidney Failure, Chronic/genetics , Male , Middle Aged , Molecular Sequence Data , Polymorphism, Genetic , Protein Isoforms/genetics , TRPP Cation Channels
8.
Biochim Biophys Acta ; 1492(2-3): 560-7, 2000 Jul 24.
Article in English | MEDLINE | ID: mdl-11004525

ABSTRACT

It is well known that breast carcinomas without estrogen receptor (ER) have a poor prognosis and do not respond to endocrine therapy. In analyzing the question of the lack of ER gene expression, we have considered the possibility that specific negative transcription factors are present in ER-negative breast cancers. Inside the P3 upstream promoter of human ER gene we identified a transcriptional regulatory sequence able to bind protein factors expressed in ER-negative MDA-MB-231 breast cancer cells. This sequence, lying between nucleotides -3258 to -3157, seems to be critical for inhibition of ER gene transcription. In fact, the selected sequence in the form of double-stranded DNA has been introduced into ER-negative breast cancer cells as 'decoy' cis elements showing the ability to remove the putative negative transcription factor(s) and to induce the reactivation of ER gene transcription. In addition, in transient transfection assays the selected sequence decreased the SV-40 promoted luciferase activity. Gel shift assays identified multiple DNA-protein interactions which specifically form in this region, and data from Southwestern experiments strongly suggested the presence of a specific protein expressed in MDA-MB-231 ER-negative, but not in MCF7 ER-positive cells.


Subject(s)
Gene Expression Regulation , Promoter Regions, Genetic/genetics , Receptors, Estrogen/genetics , Base Sequence , DNA/analysis , Gene Silencing/physiology , Humans , K562 Cells , Molecular Sequence Data , Tumor Cells, Cultured
9.
Haematologica ; 85(9): 913-21, 2000 Sep.
Article in English | MEDLINE | ID: mdl-10980628

ABSTRACT

BACKGROUND AND OBJECTIVES: The translocation t(11;14) (q13;q32), typically described in mantle cell lymphomas (MCL), has also been found in some cases of non-MCL lymphoproliferative disorders, such as splenic lymphoma with villous lymphocytes (SLVL), multiple myeloma (MM), prolymphocytic leukemia (PLL), typical and atypical chronic lymphocytic leukemia (CLL and aCLL). In order to define better the genetic features of aCLL with t(11;14), which could represent a distinct disease subset, we looked for genetic lesions in the BCL-1 locus and in BCL-2, BCL-6, c-myc and p53 genes. DESIGN AND METHODS: We investigated a panel of B-lymphoproliferative disorders with translocation t(11;14)(q13;q32) including nine aCLL, six MCL and one MM. Southern and Northern blot analysis was used to investigate DNA structure and RNA expression; SSCP and direct sequencing were used to detect and characterize p53 point mutations; cytofluorimetric analysis was used to quantify p53 protein. RESULTS: Alterations of BCL-2, BCL-6 and c-myc were not detected. Conversely, BCL-1 rearrangements were present in 4 out of 7 aCLL and in 2 out of 4 MCL. A high incidence of p53 gene alterations was found, almost equivalent in aCLL and MCL. INTERPRETATION AND CONCLUSIONS: Our results indicate that the occurrence of BCL-1 locus lesions in aCLL selected for t(11;14) is as high as in MCL. Interestingly, rearrangements in the mTC1 (minor translocation cluster 1) were only found in aCLL. Therefore, the two B-cell chronic lymphoproliferative disorders share similar molecular rearrangements and the t(11;14) identifies a subset of B-CLL sharing molecular features with MCL and characterized by aggressive clinical evolution.


Subject(s)
Chromosomes, Human, Pair 11 , Chromosomes, Human, Pair 14 , Genes, bcl-1 , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Mutation , Translocation, Genetic , Tumor Suppressor Protein p53/genetics , Adult , Aged , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Cytogenetic Analysis , Female , Gene Rearrangement , Humans , Immunophenotyping , Lymphoma, Mantle-Cell/genetics , Male , Middle Aged , Multiple Myeloma/genetics
10.
J Steroid Biochem Mol Biol ; 75(2-3): 121-8, 2000 Dec 15.
Article in English | MEDLINE | ID: mdl-11226828

ABSTRACT

It is well known that breast carcinomas without estrogen receptor (ER) have a poor prognosis and do not respond to antiestrogenic therapy. In analyzing the question of the lack of ER gene expression, we have considered the possibility to modify the ER gene expression by transfecting ER-negative breast cancer cells with a polymerase chain reaction product mimicking a putative negative regulatory region (--3258/--3157) inside the P3 ER gene promoter. Here we have demonstrated the efficacy of the selected sequence used as a decoy molecule in restoring the ER gene transcription. When this DNA was complexed and delivered by cationic liposomes (PC:DOTAP) a significant increase in the decoy effect was obtained. Breast cancer cells receiving the combination treatment responded substantially better to reactivation of quiescent ER gene than cells that had received DNA with calcium phosphate. This information may be useful for a series of in vitro transfections and also for in vivo application of the decoy strategy that is a potential therapeutic tool to control disease-related genes such as ER gene in breast cancer.


Subject(s)
Breast Neoplasms/genetics , DNA/genetics , Promoter Regions, Genetic , Receptors, Estrogen/genetics , Transfection , Blotting, Western , Cell Survival , DNA/metabolism , Drug Screening Assays, Antitumor , Fatty Acids, Monounsaturated , Female , Fluorescent Dyes , Gene Expression Regulation, Neoplastic , Genetic Therapy , Humans , Liposomes , Phosphatidylcholines , Quaternary Ammonium Compounds , Receptors, Estrogen/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, Cultured
11.
Leukemia ; 13(5): 704-7, 1999 May.
Article in English | MEDLINE | ID: mdl-10374873

ABSTRACT

A diagnosis of pro-B acute lymphoblastic leukemia (ALL) with CD15+ was made in a 42-year-old woman, 12 months after the treatment of uterine adenocarcinoma by carboplatinum, anthracyclines, etoposide and radiotherapy. Molecular cytogenetic studies revealed a karyotype with multiple chromosome changes, including the t(4;11)(q21;q23) and a 17p-chromosome, with MLL disruption and 17p13/p53 gene deletion in 86% of the cells. A p53 exon 6 mutation was documented, resulting in p53 protein stabilization, with 20% of the cells reacting with the 1801 anti-p53 monoclonal antibody. Dual-color FISH using MLL and p53 probes was performed on peripheral blood smears, providing direct evidence of the involvement of the blast cells and of the granulocytic lineage. Only a partial, shortlasting response was obtained by induction treatment, confirming that a poor prognosis is associated with therapy-related ALL with the 4;11 translocation.


Subject(s)
Chromosomes, Human, Pair 11 , Chromosomes, Human, Pair 4 , DNA-Binding Proteins/genetics , Gene Rearrangement , Genes, p53 , Mutation , Neoplasms, Second Primary/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Proto-Oncogenes , Transcription Factors , Translocation, Genetic , Adult , Female , Histone-Lysine N-Methyltransferase , Humans , Myeloid-Lymphoid Leukemia Protein
12.
Am J Kidney Dis ; 33(5): 880-5, 1999 May.
Article in English | MEDLINE | ID: mdl-10213643

ABSTRACT

The polycystic kidney disease 2 (PKD2) gene, encoding a 968-amino acid integral membrane protein with six predicted membrane-spanning domains and intracellular NH2 and COOH termini, is mutated in approximately 15% of the cases of autosomal dominant polycystic kidney disease (ADPKD), a common genetic disease frequently resulting in renal failure. For a better understanding of the cause of this disorder, we searched for mutations in the PKD2 gene in two PKD2-linked families characterized by different clinical phenotypes. A common polymorphism, a nonsense mutation, and a frameshift mutation were found. Both mutations are predicted to produce truncated proteins of 314 and 386 amino acids, arrested at the first extracellular loop of the protein. Restriction enzyme analysis of polymerase chain reaction (PCR) and reverse transcriptase (RT)-PCR products, respectively, showed that mutations cosegregated with the disease and mutated alleles were expressed at the messenger RNA level in lymphoblastoid cell lines. However, in these cells, Western blot analysis showed only PKD2 normal protein, and it was expressed at a lower level than that found in cells without the PKD2 mutation. These findings suggest that in lymphoblastoid cells, the truncated protein product of the mutant allele may not be stable.


Subject(s)
Membrane Proteins/analysis , Mutation , Polycystic Kidney, Autosomal Dominant/genetics , Aged , Alleles , Female , Humans , Male , Membrane Proteins/genetics , Middle Aged , Pedigree , Polycystic Kidney, Autosomal Dominant/blood , Restriction Mapping , Reverse Transcriptase Polymerase Chain Reaction , TRPP Cation Channels
13.
Int J Gynecol Cancer ; 9(2): 147-155, 1999 Mar.
Article in English | MEDLINE | ID: mdl-11240757

ABSTRACT

In 41 ovarian epithelial tumors (7 borderline and 34 invasive), loss of heterozygosity (LOH) of chromosomes 6q, 17q, and 18q was examined using 4 microsatellite markers: ER (6q 25-1), BRCA1 (17q21), DCC (18q21), and D18S58 (18q23). The LOH was compared with clinicopathological findings, including p53 and ER expression. In borderline tumors, LOH and p53 expression were never found, while in invasive carcinomas LOH and p53 were found in 71% and 59% of cases, respectively. In particular, in invasive carcinomas 6q LOH represented a marker distinguishing two groups of tumors; those with 6q LOH were only of serous histotype and at advanced stages (III/IV). No significant difference was found for any of genes in 5-year survival of the patients. No correlation was found between ER expression and ER LOH, as well as between biological aggressiveness and 17q and/or 18q LOH. We conclude that p53 and LOH of the investigated loci distinguish borderline from invasive ovarian carcinomas; moreover, the comparison of these results with clinicopathological parameters suggests that the presence of 6q LOH may be a factor accounting for greater biologic aggressiveness independent of the histologic subtype.

14.
J Endocrinol ; 159(3): 373-80, 1998 Dec.
Article in English | MEDLINE | ID: mdl-9834454

ABSTRACT

Evidence for the expression of the canonic androgen receptor (AR) in human adrenal cortex has not been provided so far. The aim of the present study was to demonstrate the expression of the AR gene in normal and neoplastic adrenocortical human tissues and in the human adrenocortical cancer cell line, NCI-H295, and then to evaluate the effect of dihydrotestosterone (DHT) on human adrenocortical cell growth. An AR cDNA fragment with the expected size of 262 bp was detected by using reverse transcription (RT)-PCR in normal and neoplastic adrenocortical human tissues and in the neoplastic cell line, demonstrating that the gene for AR is indeed expressed in human adrenal cells. In the human adrenocortical cancer cell line NCI-H295, DHT at physiological concentrations produced a significant reduction in cell proliferation and inhibition of colony formation in soft agar. The inhibitory effect on adrenocortical cell growth was evident after both 24 and 48 h of treatment. The antiandrogens, cyproterone acetate and hydroxyflutamide, were capable of reversing the effects exerted by DHT. The androgen-induced growth inhibitory effect was also detected in primary culture of three non-functioning adrenocortical adenomas. These findings show that the canonic AR is present in human adrenocortical cells and that androgens may have a role in the adrenal cortex by reducing cell proliferation.


Subject(s)
Adrenal Cortex/drug effects , Androgen Antagonists/pharmacology , Dihydrotestosterone/pharmacology , Receptors, Androgen/genetics , Adrenal Cortex/cytology , Adrenal Cortex/metabolism , Cell Division/drug effects , Cells, Cultured , Cyproterone Acetate/pharmacology , Female , Flutamide/analogs & derivatives , Flutamide/pharmacology , Gene Expression , Humans , Immunohistochemistry , Male , Receptors, Androgen/analysis , Receptors, Androgen/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, Cultured
15.
Cancer Genet Cytogenet ; 107(2): 137-43, 1998 Dec.
Article in English | MEDLINE | ID: mdl-9844609

ABSTRACT

We previously found that cases of typical B-chronic lymphocytic leukemia (CLL), atypical B-CLL with t(11;14) and mantle cell lymphomas characterized by rapid progression of the disease and resistance to therapy, had mutations of the TP53 gene. In this paper, abnormalities of the TP53 gene were investigated in two cases of prolymphocytic leukemia, one with t(11;14)(q13;q32), evolving from atypical CLL (patient 1), and one presenting as a de novo condition (patient 2). TP53 DNA was investigated by Southern blot and PCR-SSCP analysis, and TP53 expression was investigated by Northern blot analysis and immunocytochemistry. C-MYC and BCL-1/PRAD1 gene expression were also investigated. Restriction enzyme analysis of TP53 DNA in patient 1 showed alteration of fragments including exon I and intron I, and, in both patients, a specific loss of TP53 DNA. In patient 2, PCR direct sequencing showed in exon VII a 9 bp deletion including codons 252-254. In patient 1, TP53 RNA and protein were not found, indicating that the unusual 5' rearrangement has affected TP53 gene expression. By contrast, patient 2 exhibited detectable TP53 RNA and protein. Detectable but weak BCL-1/PRAD1 RNA was present in both patients, whereas C-MYC RNA expression was clearly present only in case 1. The presence of TP53 hemizygous mutations in both patients suggests that TP53 abnormalities may be important in the pathogenesis of prolymphocytic leukemia (PLL), and may possibly account for the frequent resistance to therapy observed in this disease.


Subject(s)
Genes, p53/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Prolymphocytic/genetics , Aged , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Exons/genetics , Female , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Leukemia, Prolymphocytic/drug therapy , Leukemia, Prolymphocytic/pathology , Male , RNA, Messenger/analysis , Tumor Suppressor Protein p53/analysis
16.
Breast Cancer Res Treat ; 49(3): 227-35, 1998 Jun.
Article in English | MEDLINE | ID: mdl-9776506

ABSTRACT

Transcriptional activity of human estrogen receptor (hER) gene was modulated by competition with double-stranded PCR-generated DNA fragments (decoys) that contain 5' upstream sequences of the hER gene. Two DNA fragments belonging to the P1 canonical promoter and the P3 distal promoter, 120 and 102 bp in size respectively, were produced by PCR and directly transfected in MCF7 breast cancer cells. After 24 hours transfection, RT-PCR analysis revealed that the 120 bp decoy significantly reduced the expression of the ER gene and estrogen responsive genes (PR and c-myc), whereas the 102 bp decoy increased the ER mRNA level. An ER unrelated PCR product, used as control, had no activity. The biological activity of these ds DNAs was related to their high stability, binding affinities, and lack of cytotoxicity. These findings suggest that such PCR product decoys may be a non-antisense tool to analyze putative regulatory sequences and to study the function of DNA-binding transcription factors.


Subject(s)
Breast Neoplasms/genetics , Gene Expression Regulation, Neoplastic , Receptors, Estrogen/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , Female , Humans , RNA, Messenger/metabolism , Tumor Cells, Cultured
17.
Exp Cell Res ; 244(1): 259-67, 1998 Oct 10.
Article in English | MEDLINE | ID: mdl-9770368

ABSTRACT

Polycystin, the PKD1 gene product mutated in autosomal dominant polycystic kidney disease, is a large membrane protein which is important in the differentiation of epithelial tubular structure. Furthermore, PKD1 mRNA is expressed in various tissues and in neoplastic cell lines particularly, suggesting that polycystin might be involved in differentiation and/or proliferation of other cell types. Therefore, in order to investigate such a possible role, polyclonal antibodies against a recombinant polycystin peptide were raised and used to study polycystin expression in human leukemia cell lines committed to differentiation. Using Western blot and laser scanning confocal microscopy analyses, we demonstrated expression of polycystin in erythroleukemia K562 cells as a membrane-associated polypeptide of approximately 450 kDa, mainly localized in cell-cell contacts. Protein size and subcellular distribution were similar to those found in the kidney epithelial KJ29 cell line. In addition, K562 cell erythroid differentiation induced by hemin was characterized by a reduction in polycystin expression, as measured by Western blot and Northern blot analyses. Cytofluorimetric analysis indicated that upon hemin treatment there was a progressive reduction in the number of polycystin-expressing cells as well as in proliferation rate. Furthermore, reduction in proliferating and polycystin-expressing cells was also observed in K562 cells after serum starvation. When serum was added to the serum-deprived cells an increase in cell number as well as in number of polycystin-positive cells was observed. In addition, polycystin, also expressed in promyelocytic leukemia HL60 cells, was downregulated when macrophage differentiation in HL60 was induced by TPA. Therefore, in these leukemic cells downregulation of polycystin appeared to be closely related to reduction in cell proliferation and to induction of differentiation. This suggests that polycystin may play a relevant role in these cell processes.


Subject(s)
Macrophages/metabolism , Macrophages/pathology , Protein Biosynthesis , Antibodies/metabolism , Cell Differentiation , Cell Division , Cell Membrane/metabolism , Down-Regulation , HL-60 Cells , Humans , K562 Cells , Polycystic Kidney, Autosomal Dominant/metabolism , Polycystic Kidney, Autosomal Dominant/pathology , Proteins/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/immunology , TRPP Cation Channels
18.
Br J Haematol ; 103(1): 189-97, 1998 Oct.
Article in English | MEDLINE | ID: mdl-9792307

ABSTRACT

To better define the role of exposure to myelotoxic agents in the genesis of myelodysplastic syndrome (MDS), we carried out (a) a case-control study for the determination of the relative risk (RR) of developing MDS, including 178 consecutive patients and 178 sex- and age-matched controls: (b) a study of clinicobiological features in MDS arising after occupational exposure to myelotoxic agents and in MDS in 'non-exposed' patients. The definition of the 'exposure' status was based on a predetermined questionnaire, with calculation of an 'exposure' index (hours/day x days/year x years). Cumulative exposure to pesticides or to organic solvents, for >2400 h, was recorded in 48 and 25 MDS patients, respectively, compared to 27 and four controls (P<0.00001; RR 3.74; 95% confidence interval 2.02-5.37). Older age and an excess of refractory anaemia with ringed sideroblasts and refractory anaemia with excess of blasts was noted among 'exposed' MDS-patients (group 1), compared to non-exposed MDS-patients (group 2). 68.3% patients in group 1 had clonal chromosome changes, compared with 43.2% patients in group 2. Complex karyotypes, -7/7q-, -5/5q-, +8, 7p and 17p aberrations were seen more frequently in group 1, whereas a normal karyotype, isolated 5q- or 20q- occurred more frequently in group 2. The association of exposure to myelotoxic agents with older age at presentation and with unfavourable chromosome changes accounted for the shorter survival observed in 'exposed' patients. These data show that occupational exposure to pesticides and organic solvents in our region resulted in an increased RR of developing MDS and that a distinct cytogenetic profile was associated with MDS in 'exposed' patients. These findings provide strong indirect evidence that these agents may play a role in the pathogenesis of MDS, preferentially targeting some of the chromosome regions which are frequently involved in therapy-related myeloid neoplasias.


Subject(s)
Carcinogens, Environmental/adverse effects , Myelodysplastic Syndromes/chemically induced , Occupational Exposure/adverse effects , Pesticides/adverse effects , Solvents/adverse effects , Adolescent , Adult , Aged , Aged, 80 and over , Case-Control Studies , Chromosome Aberrations , Humans , Karyotyping , Middle Aged , Myelodysplastic Syndromes/genetics , Myelodysplastic Syndromes/mortality , Survival Rate , Washington/epidemiology
19.
Biochem Pharmacol ; 56(6): 703-8, 1998 Sep 15.
Article in English | MEDLINE | ID: mdl-9751074

ABSTRACT

The potential use of polymerase chain reaction (PCR)-generated DNA fragments (PCR-DNAs) as pharmaceutical agents has previously been suggested, with the demonstration of the in vitro cellular internalization and biologic activity of PCR-DNA decoy molecules targeted to human estrogen receptor gene. In order to provide information on the stability of these double-stranded DNA molecules, the nuclease resistance of PCR-DNAs of different sizes was studied in different conditions and experiments. Simulating in vitro and in vivo transfection protocol, we demonstrated that PCR-DNAs exhibited good stability toward fetal bovine serum (FBS) and adult human serum nuclease digestion. In addition, when the protective activity of liposome-based formulations toward nuclease digestion was tested, it was shown that the stability of PCR-DNAs could be further increased (up to 7 days) when a liposome-mediated delivery system was employed.


Subject(s)
Cell Extracts/chemistry , DNA-Cytosine Methylases/metabolism , DNA/blood , DNA/chemistry , Polymerase Chain Reaction/methods , Adult , Breast Neoplasms/chemistry , Cations , DNA/analysis , DNA, Neoplasm/analysis , DNA, Neoplasm/chemistry , DNA, Viral/analysis , DNA, Viral/chemistry , Drug Stability , Electrophoresis, Agar Gel , Humans , Leukemia, Erythroblastic, Acute/metabolism , Liposomes , Rhabdomyosarcoma/chemistry , Tumor Cells, Cultured
20.
Int J Cancer ; 79(4): 390-5, 1998 Aug 21.
Article in English | MEDLINE | ID: mdl-9699532

ABSTRACT

The prognostic significance of chromosome 18q allelic loss was evaluated in a series of 118 patients with curatively resected TNM stage II or stage III colon cancer. Chromosome 18q status was determined on frozen tumour samples, using microsatellite markers and the polymerase chain reaction (PCR). Mean follow-up in surviving patients was 75.9 months. Chromosome 18q allelic loss was significantly related to tumour site, extramural venous invasion, flow cytometric nuclear DNA content and p53 protein expression. Patients whose tumour had no evidence of chromosome 18q allelic loss showed a better disease-free and overall survival than patients whose tumour demonstrated 18q allelic loss. When patients were stratified by tumour stage, a significant survival advantage for patients whose tumour had no allelic loss on chromosome 18q was observed in stage II as well as in stage III disease. In particular, patients with stage II disease whose tumour had no chromosome 18q allelic loss demonstrated an excellent clinical outcome, with a 5-year disease-free survival rate of 96%. In contrast, the 5-year disease-free survival rate of patients with stage II disease and chromosome 18q allelic loss was only 54%. In multivariate analysis, status of chromosome 18q was the only significant independent prognostic factor for both disease-free and overall survival. These results indicate that assessment of chromosome 18q status provides relevant prognostic information in colon cancer and might be employed in the selection of patients for adjuvant therapy.


Subject(s)
Chromosome Deletion , Chromosomes, Human, Pair 18 , Colonic Neoplasms/genetics , Adult , Aged , Aneuploidy , Colonic Neoplasms/mortality , Colonic Neoplasms/pathology , Female , Flow Cytometry , Genes, p53 , Humans , Male , Microsatellite Repeats , Middle Aged , Multivariate Analysis , Polymerase Chain Reaction , Prognosis , Time Factors
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