Molecular and biochemical mechanisms of fludarabine and cladribine resistance in a human promyelocytic cell line.

2F-Adenine arabinoside (fludarabine, Fara-A) and 2-chloro-2'-deoxyadenosine (cladribine, CdA) are nucleoside analogues with antineoplastic activity in vitro and in vivo. Lack of clinical resistance between CdA and Fara-A has been demonstrated in patients with chronic lymphocytic leukemia (G. Juliusson et al., N. Engl. J. Med., 327: 1056-1061, 1992). To clarify the differences in mechanism of resistance to CdA and Fara-A in vitro, we developed two stable, resistant cell lines, HL60/CdA and HL60/ Fara-A, by exposure to increasing concentrations of analogues over a period of 8 months. Resistant cells tolerated >8,000 and 5-fold higher concentrations of CdA and Fara-A, respectively. The specific activity of the nucleoside phosphorylating enzyme (using deoxycytidine as substrate) in cell extracts from HL60/CdA and HL60/Fara-A mutants was about 10 and 60%, respectively, compared with the parental cell line. Western blot analysis using a polyclonal antibody showed no detectable deoxycytidine kinase (dCK) protein in CdA-resistant cells, whereas in Fara-A-resistant cells, it was at the same level as in the parental cells. The mitochondrial enzyme deoxyguanosine kinase was not altered in resistant cell lines. The HL60/CdA cells showed cross-resistance to 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine, Fara-A, arabinofuranosyl cytosine, difluorodeoxyguanosine, and difluorodeoxycytidine toxicity, most likely because of the decreased phosphorylation of these analogues by dCK. Using real-time quantitative PCR, the mRNA levels of dCK and cytosolic 5'-nucleotidase (5'-NT), a major nucleoside dephosphorylating enzyme, were measured. It was shown that the dCK mRNA levels in both CdA- and Fara-A resistant cells were decreased in parallel with the activity. The expression of 5'-NT mRNA was not significantly elevated in CdA- and Fara-A resistant cells, as compared with the parental cells. Ribonucleotide reductase maintains a balanced supply of deoxynucleotide triphosphate pools in the cell and may also be a major cellular target for CdA and Fara-A nucleotides. Except for the deoxycytidine triphosphate level, the intracellular deoxynucleotide triphosphate pools were significantly higher in Fara-A-resistant cells compared with the parental cell line. This might be a consequence of mutation or altered regulation of ribonucleotide reductase activity and may explain the 2-5-fold cross-resistance to several nucleoside analogues observed with HL60/Fara-A cells. It is likely that the resistance for CdA was mainly attributable to a dCK deficiency, and Fara-A-resistant cells might have another contributing factor to the resistance beyond the dCK deficiency.

Quantitation of estrogen receptor in seventy-five specimens of breast cancer: comparison between an immunoassay (Abbott ER-EIA monoclonal) and a [3H]estradiol binding assay based on isoelectric focusing in polyacrylamide gel.

Quantitation of estrogen receptor has been performed in cytosol prepared from 75 specimens of breast cancer tissue from patients who had not received hormonal therapy. The study was performed in order to compare an immunoassay (Abbott Laboratories, North Chicago, IL) with our currently used method for estrogen receptor analysis based on isoelectric focusing of [3H]estradiol-receptor complex in polyacrylamide gels. Using linear regression analysis, a regression coefficient (slope) of 1.30 and a correlation coefficient of 0.75 were calculated. The differences in results between the two methods are probably partly explained by the fact that the ligand-based method only measures unoccupied receptor, whereas the immunoassay detects the total amount of receptor, resulting in generally slightly higher concentrations with the latter method. However, in five of 75 specimens the ligand-based method gave a considerably higher concentration of estrogen receptor. This was most probably explained by partial proteolysis resulting in the formation of receptor fragment(s), which was undetectable with the immunoassay but detectable with the ligand-based method. These observations underline the importance of careful handling of specimens during the whole immunoassay procedure.

A complex nine base pair deletion in RET exon 11 common in sporadic medullary thyroid carcinoma.

Genetic alteration of the RET proto-oncogene is associated with multiple endocrine neoplasia type 2A and 2B (MEN 2A and MEN 2B), familial medullary thyroid carcinoma (FMTC) and Hirschprung's disease. Oncogenically activated RET has also been demonstrated in sporadic medullary thyroid tumors, which in some cases show somatic missense mutations. We have recently described a complex 9 bp deletion in RET exon 11 in a single case of sporadic MTC. In order to determine the prevalence of this mutation among sporadic MTC tumors, we have now analysed 15 cases and five normal controls by PCR-based nonradioactive single-strand conformational polymorphism analysis (PCR-SSCP) and fragment size analysis of exon 11. DNA was extracted from microdissected tumor tissue or normal cells and subjected to nested PCR prior to analysis. A markedly divergent SSCP pattern and a PCR fragment 9 bp shorter than normal were demonstrated in 14 of the 15 MTC tumors. Sequencing revealed the deletion of nine bases encompassing a key cysteine at codon 634, often altered in MEN 2A. Four lymphocyte controls and normal thyroid tissue from one patient failed to show the deletion. Several factors in the DNA sequence environment immediately surrounding the deletions, including an extended inverted repeat, several direct repeats and a so-called symmetric element suggest that the deletional events may be non-random.

Epstein-Barr virus (EBV) can immortalize B-cll cells activated by cytokines.

B-type of chronic lymphocytic leukemia (B-CLL) cells are inert to the potent transforming action of Epstein-Barr virus (EBV). The mitogenic action of Staphylococcus aureus Cowan I (SAC), MP6-thioredoxin, and interleukin 2 (IL-2), agents previously shown to induce proliferation in normal as well as in B-CLL cells, lifted this block, and EBV-positive cell lines could be established. It was not possible to establish cell lines of leukemic origin from cultures that were incubated with EBV alone or cytokine mix alone. CLL-cells infected with EBV only, expressed the viral nuclear antigen complex (EBNA), but not the viral latent membrane protein (LMP). They were not activated as measured by cell size and 3H-thymidine incorporation. In contrast, cells incubated with EBV and cytokine mix expressed both EBNA and LMP in parallel with enlargement and increased 3H-thymidine incorporation. These results emphasize that LMP expression is a prerequisite for growth transformation and immortalization and that cytokine activation signals are required for its expression in B-CLLs. Cells incubated with SAC/MP6-thioredoxin/IL-2 did not express any of the viral antigens, but were activated with regard to the mentioned parameters. Nine cell lines were established from six patients. From each of the three patients, we obtained 'twin'-pair lines: one corresponding to the malignant cell and the other to a normal B-lymphoblastoid cell. Thus, malignant and normal B-cell counterparts, from the very same donor, are at hand for comparative studies. The cell lines have been carried out for more than 12 months in culture. We conclude that B-CLL that are refractory to EBV-transformation can be rendered susceptible through in vitro cytokine activation.

Diadenosine pentaphosphate modulates glomerular arteriolar tone and glomerular filtration rate.

INTRODUCTION: Mechanisms and participating substances involved in the reduction of glomerular filtration (GFR) in contrast-induced acute kidney injury (CI-AKI) are still matter of debate. We hypothesized that diadenosine polyphosphates are released by the action of contrast media on tubular cells and may act on glomerular arterioles and reduce GFR. METHODS: Freshly isolated rat tubules were treated with the contrast medium iodixanol (47 mg iodine per mL) at 37 °C for 20 min. The content of Apn A (n = 3-6) in the supernatant of treated tubules and in the plasma of healthy persons and patients with AKI was analysed using reversed-phase chromatography, affinity chromatography and mass spectrometry. GFR was obtained in conscious mice by inulin clearance. Concentration response curves for Apn A (n = 3-6, 10(-12) -10(-5)  mol L(-1) ) were measured in isolated perfused glomerular arterioles. RESULTS: Iodixanol treatment of tubules significantly increased the concentration of Apn A (n = 3-5) in the supernatant. Ap6 A was below the detection limit. AKI patient shows higher concentrations of Apn A compared to healthy. Application of Ap5 A significantly reduced the GFR in conscious mice. Ap5 A reduced afferent arteriolar diameters, but did not influence efferent arterioles. The constrictor effect on afferent arterioles was strong immediately after application, but weakened with time. Then, non-selective P2 inhibitor suramin blocked the Ap5 A-induced constriction. CONCLUSION: The data suggest that Ap5 A plays a role in the pathophysiology of CI-AKI. We show a contrast media-induced release of Ap5 A from tubules, which might increase afferent arteriolar resistance and reduce the GFR.

Thyroid-infiltrating T lymphocyte subsets in Hashimoto's thyroiditis.

In Hashimoto's thyroiditis, the thyroid gland is infiltrated with immunocompetent lymphocytes. In this study we have used the fine needle aspiration technique to obtain thyroid-infiltrating lymphocytes from 11 HT patients for surface marker studies. The cells were characterized using conventional T and B cell markers as well as monoclonal antibodies (OKT) to different T cell subsets in a microscale immunofluorescence assay. We observed a relative decrease in intrathyroidal suppressor phenotype T (OKT 8+) cell numbers compared with peripheral blood (13% vs. 26%; P less than 0.01 by Wilcoxon signed rank test). This resulted in an increased helper to suppressor T (OKT 4+/OKT 8+) cell ratio (4.7 vs. 2.1; P less than 0.01). Within the gland, a significant local accumulation of B cells was also registered (27% vs. 12%; P less than 0.01). As to circulating lymphocyte subsets, no differences were found between 18 Hashimoto's thyroiditis patients and 26 normal subjects. Our results are compatible with a local synthesis of thyroid-directed antibodies and emphasize the importance of studying the local immunity in organ-specific autoimmune disease.

Intrathyroidal and circulating lymphocyte subsets in different stages of autoimmune postpartum thyroiditis.

Postpartum thyroiditis (PPT) is a reversible form of lymphocytic thyroiditis which has been attributed to an aggravation of preexisting subclinical autoimmune thyroiditis. In this study no differences in circulating lymphocyte subsets were found between 9 thyrotoxic and 18 hypothyroid PPT patients and normal subjects. We obtained sufficient numbers of thyroid-infiltrating lymphocytes for surface marker characterization in 3 women in the thyrotoxic phase and in 10 women in the hypothyroid phase of PPT. Cells were identified by conventional T and B cell markers as well as by monoclonal antibodies (OKT) directed against different T cell subsets in a microscale immunofluorescence assay. In the hypothyroid patients a relative accumulation of B cells (31% vs. 17%; P less than 0.01 by the Wilcoxon signed rank test) was found within the thyroid when compared to peripheral blood. A relative decrease in intrathyroidal supressor-cytotoxic (OKT 8+) T cells (19% vs. 28%; P less than 0.01) resulted in an increased intrathyroidal helper to suppressor-cytotoxic (OKT 4+/OKT 8+) T cell ratio (3.0 vs. 2.0; P less than 0.01). Intrathyroidal lymphocyte subsets in the thyrotoxic patients were comparable to those in the hypothyroid patients. These findings, which are similar to those we previously obtained in patients with chronic Hashimoto's thyroiditis, may indicate that local synthesis of thyroid-directed autoantibodies is of primary importance in all stages of autoimmune thyroiditis.

Clonality analysis of cervical cancer on microdissected archival materials by PCR-based X-chromosome inactivation approach.

Clonality of invasive human cervical cancer was assessed in microdissected archival formaldehyde-fixed, paraffin-embedded tissues by PCR-based analysis of X-chromosome inactivation of the androgen receptor gene. In 18 informative cases, cancers from 12 cases including 2 early-invasive cancers were scored as monoclonal. Among them, 8 cases had the combination of random X-chromosome inactivation in normal cervical tissue and non-random in the cancer and in other 4 cases normal and cancer tissue were both non-random but had discordant X-chromosome inactivation. Six cases showed that the non-random inactivation affected the same allele in cancer and normal tissue and thus were considered to be inconclusive. The results suggest the monoclonal origin of cervical cancer and indicate that genetic events are critical in transition of pre-malignant epithelia to invasive cancer in cervical carcinogenesis. Finding of monoclonal early invasive cancer also argues that monoclonality of human tumors is not a late event due to clonal competition or selection. X-chromosome inactivation patterns in normal cervical epithelia and tissues were analyzed in 21 informative cases. When a mixture of normal stroma and epithelium was analyzed, 38% (8/21) of cases showed non-random inactivation pattern, whereas using pure epithelium it was 64% (11/17). The X-chromosome inactivation pattern between mixed epithelium/stroma and matched epithelium differed in 2/8 cases. These results point to a certain amount of overlooked source of error in X-inactivation-based tumor clonality analysis in which peripheral blood monocytes were chosen as control and strongly recommend that control tissues which share close histological origin with analyzed tumors should be selected in any clonality study of human neoplasm. Post-embryological mechanisms of clonal patch in normal cervical epithelia are also discussed.

Molecular pathology in the diagnosis of hematologic neoplasia. Review article.

Over the past decade molecular genetic methods have played an increasingly important role in the diagnosis of hematologic malignancies. Moreover, they have provided a tool to analyze many of the non-random cytogenetic anomalies associated with hematologic neoplasias, contributing considerably to our understanding of several of those diseases, and to improving diagnostic accuracy. The rapid development of molecular genetics progressively allows the replacement of time-consuming and technically demanding procedures. Even more relevant are the new clinical applications that already include the search for valuable prognostic information and ways of evaluating minimal residual disease or recognizing early relapsing disease. This paper is a critical but necessarily simplified overview of the main contributions of molecular genetics to the field of hematopathology. We discuss the information provided by several molecular methods within different clinical contexts, covering common problems in diagnostic pathology as well as prognostic evaluation and therapy monitoring.

Rapid test for identification of a human papillomavirus 16 E6 L83V variant.

A variant of human papillomavirus (HPV) 16 has been shown recently to be more prevalent in invasive cervical carcinoma than in preinvasive lesions. This HPV 16 variant possesses a common mutation (T to G) in nucleotide 350 (codon 83) of the E6 gene, resulting in an amino acid shift, L83V, in the E6 protein. This mutation was believed to signify preinvasive cervical lesions with a high probability of progression to invasive carcinoma. The purpose of the present investigation is to describe a rapid method for the detection of this variant HPV 16, E6 (L83V). Paraffin blocks of 18 gynecologic biopsy specimens were collected, all displaying the morphology of cervical intraepithelial neoplasia (CIN I-III) and a positive HPV 16 test. Sections from these blocks were used for DNA extraction. A DNA sequence of the E6 gene containing 176 bases (including codon 83) was amplified by polymerase chain reaction (PCR) and analyzed by non-radioactive single-strand conformational polymorphism (SSCP) analysis. A divergent SSCP pattern was observed in 7 of the HPV 16 positive biopsy specimens. A DNA sequence analysis of the PCR products revealed the conversion of Leu to Val in codon 83 of the E6 gene, correlating to the divergent band pattern. This PCR-SSCP method can be used to test for HPV 16 in women who are at serious risk of developing invasive cervical carcinoma.

Polymerase chain reaction-single-strand conformational polymorphism analysis of antigen receptor rearrangements in monitoring therapeutic effect in childhood ALL.

The rearrangement of the immunoglobulin heavy chain (IgH) genes can be used as a marker of cell lineage and clonality. The polymerase chain reaction (PCR) technique using consensus primers for the IgH gene was used for remission and minimal residual disease (MRD) analysis in the follow-up of childhood acute lymphoblastic leukemia (ALL) of B-cell lineage. Single-strand conformational polymorphism (SSCP) was used to distinguish the specific clonal amplicons from the background. The Authors found that, in a series of 22 patients followed-up for 5.3 to 11.1 years, the PCR-SSCP technique could detect at least one rearrangement at initial diagnosis in 21 (95%). All patients who remained in continuous complete remission were PCR-SSCP negative at remission controls. Ten of the 22 patients had one or more bone marrow relapses. The PCR-SSCP method demonstrated MRD in three of them. In 6 of the 7 (86%) of patients with disease recurrence from whom samples were taken within 6 months before a clinically overt relapse, PCR-SSCP became positive. The Authors conclude that PCR-SSCP of a rearrangement marker might have a role as a convenient technique for monitoring emerging relapse. It may also detect unrelated clones or ongoing secondary recombination events during progression. However, PCR-SSCP is not sensitive enough to detect MRD in all patients in whom disease will later recur.

Comparative analysis of detection systems for evaluation of PCR amplified immunoglobulin heavy-chain gene rearrangements.

Four different detection systems were compared for evaluation of polymerase chain reaction (PCR) amplified immunoglobulin heavy-chain gene rearrangements in acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma (NHL) of B-cell lineage. In 63.0% of the fragments detected by ethidium bromide stained agarose gel electrophoresis (Agarose-EtBr) the sensitivity was insufficient to separate the specific clonal population from the background of normal B cells. Using polyacrylamide gel electrophoresis (PAGE), PAGE combined with single-strand conformation polymorphism (PAGE-SSCP) and PhastGel-SSCP (Phast-SSCP) analysis with silver staining, the resolution was improved and the majority of the inconclusive amplicons were elucidated. However, Phast-SSCP displayed a slightly higher detection level compared to PAGE and PAGE-SSCP. According to our findings PAGE-SSCP and Phast-SSCP were superior to agarose-EtBr and PAGE in detecting new emerging clones and clonal evolution.

Nonradioisotopic detection and typing of human papillomaviruses by use of polymerase chain reaction and single-strand conformation polymorphism.

The polymerase chain reaction (PCR), used to detect human papillomavirus (HPV), is finding increasing applications in clinical laboratories. The standard method of analysis to detect amplified PCR products is ethidium bromide gel electrophoresis combined with labor intensive blot hybridization. In this study, we describe single-strand conformation polymorphism (SSCP) to detect and genotype simultaneously general primer GP5+/GP6+ amplified HPV DNA using semiautomated electrophoresis on polyacrylamide gels (PAGE) combined with sensitive silver staining. To establish a standard for the band patterns of the various HPV types, we used HPV plasmid DNA, which allowed us to distinguish HPV 6, 11, 16, 18, 31, 33, 35, 45, 51, 52, 56, and 58, covering the most frequently recognized types. All the types tested are separated from each other, demonstrating diverse band patterns, HPV 16 being the most distinct. We also investigated PCR-SSCP for HPV detection and typing of 86 cervical biopsies diagnosed as cervical intraepithelial neoplasia (CIN) I-III and known to be HPV positive by PCR-slot blot hybridization and in situ hybridization. The correlation with SSCP was 91% for in situ hybridization and 98% for PCR-slot blot hybridization. SSCP is reproducible and specific. Its sensitivity is comparable to slot-blot hybridization. The interval to SSCP is approximately 2 h after PCR compared with several days' work when using conventional blot hybridization. We concluded that SSCP may be more advantageous than other PCR-based typing technologies.

Remarks upon the effect of temperature and dextran coated charcoal on buffer pH in steroid receptor assays.

Phosphate and TRIS buffers of low molarity commonly used for steroid receptor assays were studied for their pH stability. Increased temperature from 0-37 degrees C lowers the pH of TRIS buffer, while phosphate buffer remains approximately constant. Addition of dextran and charcoal may change the pH unacceptably, depending on the charcoal quality.

Deletion of chromosome 3p is an early event in malignant progression of cervical cancer.

BACKGROUND: Loss of chromosome 3p has been reported to be a common genetic alteration in certain types of tumors as well as in cervical cancer. To understand its role in multistep cervical carcinogenesis, we analysed the allelic loss of this chromosome region in early pre-invasive cervical neoplastic lesions. MATERIALS AND METHODS: 49 cases comprising two types of pre-invasive lesions (without and with coexisting invasive cancer) were selected. Chromosome 3p deletios in selected neoplastic lesions were detected by PCR-RFLP combined with morphologically guided microdissection technique for DNA preparation. RESULTS: DNA samples from 40 cases out of 49 were successfully amplified and found to be heterozygotes at least in one chosen chromosome marker. Of 19 cases without coexisting cancer, LOH was detected in 4 cases(21%) and 3/17(18%) precancerous lesions (1 moderate dysplasia, 2 severe dysplasias) and 1/5(20%) carcinomas-in-situ. A significantly higher rate of allelic loss was observed in pre-invasive lesions adjacent to invasive cancer, showing 29%(4/14) in precancerous lesion and 67%(2/3) in carcinoma-in-situ. Analysing the invasive cancer and synchronous pre-invasive neoplasia, LOH was found to occur at early precursor stage in the majority of cases(5/7). The most frequently lost locus in cervical cancer is the chromosome regions detected by marker D3S30(60%) and D3F15S2(47%), suggesting a candidate tumor suppressor gene, which may play a role in cervical carcinogenesis, is harboured on this chromosome region. CONCLUSIONS: Our results confirm the high frequency of chromosome 3p allelic loss in cervical cancer and suggest that this genetic alteration is an early event in the development of cervical cancer and may potentially serve as a marker of risk for progression of premalignant lesions to invasive cancer.

Variation of pH of mammary tumour cytosols: potential influence on steroid receptor assay.

Mammary tumour cytosols prepared for steroid receptor assay showed pH values above or below 7.3-7.5 in 50% of the samples. An increase of 0.6 pH units was observed after the addition of dextran coated charcoal. To test the sensitivity of the assay systems to pH changes, breast cancer cytosols were adjusted to pH between 6.0-9.0 before incubation with ligand at 0 degrees C. ER was then separated by isoelectric focusing in a pH gradient between 9.5-3.5 in a routine assay including a dextran coated charcoal step before focusing. PgR was assayed by electrofocusing as well as by a conventional DCC method and Scatchard plot. Unadjusted control cytosols were run in parallel. At pH above or below 7.0-8.0 recovery of receptor was reduced, the loss being most prominent at low pH. At low pH levels Scatchard plots appeared to be of curvo-linear shape. The effect of low intracellular tumour pH on receptor-ligand affinity in vitro may be of great importance as well in vivo, and should be taken into consideration in the therapeutic situation.

Increased recovery of estrogen receptor in crude homogenates of breast cancer biopsies. A preliminary report.

Crude homogenates and cytosols of 31 breast cancer biopsies were analyzed for estrogen receptor, using the ER-EIA monoclonal assay (ABBOTT Laboratories). The recovery of the receptor was significantly higher in all homogenates than in their corresponding cytosols, in six of which the receptor was below the level of detection. The range of the ratio between homogenate and cytosol concentrations was 0.9-8.1 for 25 positive biopsies. If the recent hypothesis of nuclear localization of the receptor is accepted, the values obtained with crude homogenates would more correctly reflect the true concentration of the receptor in the cells.

Pitfalls of in situ polymerase chain reaction (PCR) using direct incorporation of labelled nucleotides.

False positivity is reported of in situ PCR reactions in a direct incorporation assay with digoxigenin-labelled dUTP. It is recommended that in situ hybridization with specific labelled probe replaces the direct incorporation method for the detection of in situ PCR amplicon.

Detection of single HPV copies in SiHa cells by in situ polymerase chain reaction (in situ PCR) combined with immunoperoxidase and immunogold-silver staining (IGSS) techniques.

Detection of HPV by means of in situ hybridization techniques may often present problems if the number of HPV copies is too small, especially when using nonradioactive detection systems. A new way of amplifying extremely small amounts of virus DNA copies is the polymerase chain reaction (PCR), which is mostly used in vitro. In this study, we present a method for in situ PCR combined with immunogold-silver staining (IGSS) and immunoperoxidase (IMP) methods which allows the detection of single copies of HPV-DNA in SiHa cells. This method may have wide applications in routine diagnostic histopathology.