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At least 1,225 records · Page 68Linked to original sources
PubMed2003

Tenascin-C upregulates matrix metalloproteinase-9 in breast cancer cells: direct and synergistic effects with transforming growth factor beta1.

Tenascin-C (TN-C) and matrix metalloproteinases (MMPs) are highly expressed in cancer tissues and probably promote cell migration during cancer progression. TN-C and MMPs are often co-localized in areas of active tissue remodeling in pathologic conditions, suggesting reciprocal regulation. To investigate whether TN-C regulates MMPs expression in cancer cells, we first exposed mammary cancer cells derived from TN-C-deficient mice to TN-C and examined MMPs expression. TN-C was then compared with fibronectin (FN), laminin (LN), basic fibroblast growth factor (b-FGF) and transforming growth factor-beta1 (TGF-beta1). Results of endpoint RT-PCR, quantitative real-time RT-PCR and gelatin zymography demonstrated that TN-C, strongly and dose dependently, upregulates MMP-9 expression in murine mammary cancer cells. TN-C weakly induced MMP-2, MMP-3 and MMP-13. FN and LN induced MMP-9 to lesser extents compared with TN-C. b-FGF had no effect on MMP-9 expression. TGF-beta1 induced MMP-9 expression in a dose-dependent manner, and this induction was significantly enhanced by addition of TN-C. TN-C and TGF-beta1 also upregulated MMP-9 expression in the human breast cancer cell line MDA-MB-231. Neutralization with specific anti-TGF-beta1 antibody showed decreased expression of MMP-9, indicating that TGF-beta controls the baseline MMP-9 expression by a direct autocrine mechanism. Under neutralization of TGF-beta, addition of TN-C still upregulated MMP-9. Conversely, neutralization of endogenous TN-C (in a TN-C-positive mammary cancer cell line) downregulated TGF-beta-induced MMP-9 expression. Thus, TN-C induces MMP-9 expression directly and by collaboration with TGF-beta. These findings reveal a novel role of TN-C in breast cancer progression.

Animals↗
PubMed2005

Vasoactive intestinal peptide induces neuroendocrine differentiation in the LNCaP prostate cancer cell line through PKA, ERK, and PI3K.

BACKGROUND: Neuroendocrine (NE) differentiation in prostate cancer has been correlated with unfavorable clinical outcome. The mechanisms by which prostate cancer acquires NE properties are poorly understood, but several signaling pathways have been proposed. We have previously observed that vasoactive intestinal peptide (VIP) stimulates cAMP production mainly through VPAC(1) receptor, inducing NE differentiation in LNCaP cells. The aim of this study was to analyze the mechanisms involved in this process. METHODS: Reverse transcriptase (RT)-polymerase chain reaction (PCR), quantitative real-time RT-PCR, Western blotting, and immunocytochemistry were performed. RESULTS: LNCaP cells produce VIP, as demonstrated by RT-PCR and immunocytochemistry. VIP induced NE differentiation of LNCaP cells at a time as short as 1 hr of treatment, and the same occurred with the expression and secretion of neuronal-specific enolase (NSE, a NE differentiation marker). These effects were faster than those exerted by serum-deprivation. VIP induced extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation and NE differentiation by PKA-dependent and independent pathways, since the PKA inhibitor H89 partially blocked VIP-induced NE differentiation and did not affect ERK1/2 phosphorylation. mitogen-activated protein kinase kinase (MEK) and phosphoinositide 3-kinase (PI3K) appear to be also involved since the inhibitors PD98059 and wortmannin abolished ERK1/2 phosphorylation and decreased NE differentiation induced by VIP. Moreover, VIP activated Ras suggesting the involvement of a Ras-dependent pathway. CONCLUSIONS: VIP behaves as autocrine/paracrine factor in LNCaP cells by inducing NE differentiation through PKA, ERK1/2, and PI3K.

Carcinoma, Neuroendocrine↗
PubMed2002

Accurate quantitative RT-PCR for relative expression of Slo splice variants.

Much interest has been shown in the use of multi-template reverse transcription-polymerase chain reaction (RT-PCR) as a quantitative instrument for low-abundance mRNAs. A desire to achieve finely-graded quantification of the stress- and hormone-related regulation of one splicing decision in an ion channel gene motivated us to test the reliability of simultaneous amplification of two splice variants with one pair of flanking constitutive primers. Unexpectedly indiscriminate heteroduplexing between the two amplification products, despite a large length difference, and their tight comigration with one homoduplex, mandated a rigorously-denaturing electrophoresis protocol. Conveniently, a new fluorescent dye with high affinity for single-stranded DNA has become available. Though the dye has a good dynamic range, we found that dye and gel saturation compounded by the length difference between products introduced an asymmetrical error into the calculation of relative abundance. Avoiding several pitfalls, dye calibration could be used to correct the error. We also found that differences in the amplification efficiency of the two templates were not constant, but dependent on the initial template ratio, requiring a non-linear correction. Together these improvements gave us very consistent quantitative results, and thus advance our analysis of hormonal mechanisms underlying the regulation of alternative splicing of an ion channel critically involved in stress responses.

Animals↗
PubMed2002

Epstein-Barr virus and post-transplant lymphoproliferative disease.

There is convincing evidence that Epstein-Barr virus (EBV) is associated with post-transplant lymphoproliferative disease (PTLD). Primary EBV infection following transplantation occurs in as many as 90% of cases of PTLD in children and pretransplant EBV seronegativity is a recognized risk factor for developing PTLD. Other risk factors include young age at the time of transplant, the type of transplant that the recipient receives and the type and intensity of immunosuppression. The clinical presentation is often nonspecific and tissue biopsy is necessary to establish the diagnosis. There appears to be a correlation between PTLD and EBV viral load measured by polymerase chain reaction (PCR) of the peripheral blood and quantitative PCR may be a useful guide in the management of PTLD. Antiviral drugs and cytomegalovirus-immunoglobulin G may have a role in preventing PTLD. Because PTLD results from functional over-immunosuppression, the initial treatment is reduction of immunosuppression. Antiviral agents, interferon, immuno-based monoclonal therapy, cell-based therapy and chemotherapy also have a potential role in treating this disorder. At the present time there is no standardized approach to the evaluation and treatment of PTLD.

Child↗
PubMed2005

Molecular and pharmacological characteristics of transient voltage-dependent K+ currents in cultured human pulmonary arterial smooth muscle cells.

The A-type voltage-dependent K(+) current (I(A)) has been identified in several types of smooth muscle cells including the pulmonary artery (PA), but little is known about the pharmacological and molecular characteristics of I(A) in human pulmonary arterial smooth muscle cells (hPASMCs). We investigated I(A) expressed in cultured PASMCs isolated from the human main pulmonary artery, using patch-clamp techniques, reverse transcriptase-polymerase chain reaction (RT-PCR), quantitative real-time RT-PCR and immunocytochemical studies. With high EGTA and ATP in the pipette, the outward currents were dominated by a transient K(+) current (I(A)), followed by a relatively small sustained outward current (I(K)). I(A) was inhibited by 4-aminopyridine (4-AP) concentration-dependently, and could be separated pharmacologically into two components by tetraethylammonium (TEA) sensitivity. A component was sensitive to TEA, and the second component was insensitive to TEA. I(A) was inhibited by blood depressing substrate (BDS)-II, a specific blocker of K(V)3.4 subunit, and phrixotoxin-II, a specific blocker of K(V)4.2 and 4.3. Flecainide inhibited I(A) concentration-dependently, but it inhibited it preferentially in the presence of TEA (TEA-insensitive I(A)). Systematic screening of expression of K(V) genes using RT-PCR showed the definite presence of transcripts of the I(A)-encoding genes for K(V)3.4, K(V)4.1, K(V)4.2 and K(V)4.3 as well as the I(K)-encoding genes for K(V)1.1, K(V)1.5 and K(V)2.1. The real-time RT-PCR analysis showed that the relative abundance of the encoding genes of I(A) alpha-subunit and K(V) channel-interacting proteins (KChIPs) was K(V)4.2 > K(V)3.4 > K(V)4.3 (long) > K(V)4.1, and KChIP3 >> KChIP2, respectively. The presence of K(V)3.4, K(V)4.2 and K(V)4.3 proteins was also demonstrated by immunocytochemical studies, and confirmed by immunohistochemical staining using intact human PA sections. These results suggest that I(A) in cultured hPASMCs consists of two kinetically and pharmacologically distinct components, probably K(V)3.4 and K(V)4 channels.

Calcium-Binding Proteins↗
PubMed2002

Up-regulation of tissue-type transglutaminase after traumatic brain injury.

Tissue-type transglutaminase (tTG, EC 2.3.2.13) has been implicated in various disease paradigms including neurodegenerative disease. In these studies, tTG induction after traumatic brain injury was studied using a rat cortical impact model. Using western blots, two forms of tTG protein expression were identified--an approximately 79-kDa primary form (tTG-L) and a less abundant approximately 70-kDa form (tTG-S). Both forms of tTG protein were elevated after injury. In ipsilateral cortex, peak induction of tTG-L protein [561% +/- 80% of control (n = 5)] was observed five days after injury, with expression remaining elevated after two weeks. Peak induction of tTG-S protein [302% +/- 81% of control (n = 5)] was observed three days after injury. Lesser tTG protein induction was observed in hippocampus. Northern blot analysis demonstrated two tTG transcripts in the ipsilateral cortex with peak induction of tTG-L mRNA three days after injury. However, tTG-S mRNA was not identified in control samples and only faintly detected in injured tissue. To facilitate analysis of low abundance transcripts in smaller tissue samples, a semiquantitative real-time PCR strategy was used. Semi-quantitative PCR analysis of tTG-L mRNA induction in ipsilateral cortex (peak after three days; 414% +/- 21% of control, n = 3) confirmed tTG-L mRNA induction determined by northern blot (410% of control).

Animals↗
PubMed2003

The loss of retinoic acid receptor alpha, beta and alcohol dehydrogenase3 expression in non-small cell lung cancer.

OBJECTIVE: The reason for unsuccessful retinoid chemoprevention of lung cancer is obscure. Therefore we examined the retinoid-related receptor expression and the tissue distribution as well as the possible interaction between retinoid-related receptor expression and cigarette smoke. METHODOLOGY: Using semiquantitative conventional reverse transcriptase polymerase chain reaction (RT-PCR), quantitative real-time RT-PCR, in situ hybridization and immunostaining, we evaluated the expression and the distribution of retinoic acid receptor alpha, beta, gamma, retinoid X receptor alpha, beta, gamma, thyroid hormone receptor alpha, beta, and alcohol dehydrogenase3 (a well-characterized retinoic acid responsive gene) in malignant and non-malignant tissues obtained from patients with non-small cell lung cancer. We also assessed the response of the bronchial epithelial cell line, BEAS-2B, following exposure to cigarette smoke extract. RESULTS: Compared with non-malignant tissues, malignant tissues had a 45% (P < 0.02) reducion in retinoic acid receptor alpha and beta expression, while other receptors were not affected. The expression of the alcohol dehydrogenase3 gene in malignant tissue was 50% of that observed in non-malignant tissue (P < 0.05). There was a significant correlation between the retinoic acid receptor beta-mRNA level and the alcohol dehydrogenase3-mRNA level (r = 0.52, P < 0.05). In situ hybridization and immunostaining demonstrated that retinoic acid receptor alpha and beta were localized in the airway and alveolar epithelial cells and their expression was diminished to 30% in malignant tissues compared to non-malignant tissues (P < 0.05). Interestingly, cigarette smoke extract decreased retinoic acid receptor beta mRNA in BEAS-2B cells (P < 0.05). CONCLUSIONS: The present findings suggest a possible association of the loss of retinoic acid receptor alpha, beta and alcohol dehydrogenase3 with lung carcinogenesis.

Adult↗
PubMed2004

Basic fibroblast growth factor expression in isolated small human ovarian follicles.

Basic fibroblast growth factor (bFGF) is involved in cell proliferation, differentiation, and angiogenesis. It has long been known that bFGF acts as a powerful mitogen for various mammalian granulosa cells in culture. To investigate the possible involvement of bFGF expression in follicle initiation and growth in vivo, we performed nested RT-PCR on ovarian cortical biopsies and quantitative PCR on human follicle populations isolated by laser capture microdissection. Using morphological criteria, follicles were characterized as putative non-growing, primary, or small secondary. RNA was extracted from samples, reverse-transcribed, and relative gene expression levels determined with TaqMan real-time PCR, using 18S rRNA as the endogenous control. Results confirmed bFGF expression in human adult ovarian cortex, and in the isolated follicles a down-regulation of bFGF mRNA was evident as small follicles develop. This study demonstrates a possible relationship between bFGF mRNA expression and follicle development.

Adult↗
PubMed2003

Identification and characterization of avian retroviruses in chicken embryo-derived yellow fever vaccines: investigation of transmission to vaccine recipients.

All currently licensed yellow fever (YF) vaccines are propagated in chicken embryos. Recent studies of chick cell-derived measles and mumps vaccines show evidence of two types of retrovirus particles, the endogenous avian retrovirus (EAV) and the endogenous avian leukosis virus (ALV-E), which originate from the chicken embryonic fibroblast substrates. In this study, we investigated substrate-derived avian retrovirus contamination in YF vaccines currently produced by three manufacturers (YF-vax [Connaught Laboratories], Stamaril [Aventis], and YF-FIOCRUZ [FIOCRUZ-Bio-Manguinhos]). Testing for reverse transcriptase (RT) activity was not possible because of assay inhibition. However, Western blot analysis of virus pellets with anti-ALV RT antiserum detected three distinct RT proteins in all vaccines, indicating that more than one source is responsible for the RTs present in the vaccines. PCR analysis of both chicken substrate DNA and particle-associated RNA from the YF vaccines showed no evidence of the long terminal repeat sequences of exogenous ALV subgroups A to D in any of the vaccines. In contrast, both ALV-E and EAV particle-associated RNA were detected at equivalent titers in each vaccine by RT-PCR. Quantitative real-time RT-PCR revealed 61,600, 348,000, and 1,665,000 ALV-E RNA copies per dose of Stamaril, YF-FIOCRUZ, and YF-vax vaccines, respectively. ev locus-specific PCR testing of the vaccine-associated chicken substrate DNA was positive both for the nondefective ev-12 locus in two vaccines and for the defective ev-1 locus in all three vaccines. Both intact and ev-1 pol sequences were also identified in the particle-associated RNA. To investigate the risks of transmission, serum samples from 43 YF vaccine recipients were studied. None of the samples were seropositive by an ALV-E-based Western blot assay or had detectable EAV or ALV-E RNA sequences by RT-PCR. YF vaccines produced by the three manufacturers all have particles containing EAV genomes and various levels of defective or nondefective ALV-E sequences. The absence of evidence of infection with ALV-E or EAV in 43 YF vaccine recipients suggests low risks for transmission of these viruses, further supporting the safety of these vaccines.

Animals↗
PubMed2003

Expression of a novel C-type lectin in the mouse vagina.

Estrogens regulate the proliferation and differentiation of mouse vaginal epithelial cells. However, the molecular mechanisms underlying estrogen-induced changes have not been elucidated. The goal of this study was to identify estrogen-responsive genes related to the proliferation and differentiation of mouse vaginal epithelial cells. We used differential display to reveal specific genes regulated by estrogens and identified a transcript that was designated DDV10. DDV10 encodes a membrane protein with a C-type lectin domain in the carboxyl-terminal region; thus, we inferred that it belongs to the C-type lectin family. We analyzed the temporal and spatial expression of DDV10 using RT-PCR, quantitative real-time RT-PCR, and in situ hybridization. Ovariectomy decreased DDV10 mRNA levels, whereas 17beta-estradiol treatment increased expression of DDV10 mRNA in vaginas of ovariectomized mice. DDV10 mRNA was first detected between 20 and 30 d after birth and was found in eye, tongue, stomach, and stratified and cornified vaginal epithelial cells, but not in stromal cells or uterus. DDV10 transcripts were not detected in vaginas of estrogen receptor alpha knockout mice. Taken together, these data suggest that DDV10 encodes a novel, 17beta-estradiol-regulated, C-type lectin in the mouse vagina. DDV10 may play a role in the stratification and/or cornification of epithelial cells during differentiation.

Animals↗
PubMed2004

Expression analysis of genes identified by molecular profiling of VGP melanomas and MGP melanoma-positive lymph nodes.

Microarray profiling is a powerful approach to establish gene expression patterns for different histopathological stages of a malignancy, and at the same time, to identify individual genes that may have important functions in the early and/or advanced stages of a neoplasm. To identify genes that hitherto have not been shown to be expressed or play a role in advanced-stage melanomas, we conducted microarray analyses with RNAs from primary melanoma and melanoma-positive lymph node specimens. Using RT-PCR, quantitative, real-time RT-PCR, and fluorochrome oligonucleotide-based optical imaging, we established the level and pattern of expression of five of the identified known genes [Suppression of Tumorigenicity 13 (ST13), Cystatin 8 (CST-8), Dyskeratosis Congentia 1 (DKC1), Neuroendocrine Secretory Protein 55 (NESP55), Niemann-Pick Disease, type C2 (NP-C2)], and a gene with unknown function (16.7 kD Hypothetical Protein) in benign and atypical nevocytic lesions, advanced-stage melanomas, and melanoma-positive lymph nodes.

Base Sequence↗
PubMed2001

[Diagnosis of H. pylori infection by PCR].

Because of its high sensitivity, polymerase chain reaction (PCR) method can be used to detect the presence of very few numbers of H. pylori organisms in gastric biopsy materials or gastric juice samples. PCR has also been applied to the detection of H. pylori organisms in the oral cavity, in stools, and in the environment. RT-PCR is useful to study the expression of H. pylori pathogenic genes and gene expression of gastric mucosal cells in response to H. pylori infection. Other PCR-based techniques, such as PCR-RFLP or real-time quantitative PCR, are now providing important information on H. pylori and pathophysiology of H. pylori infection.

Gastric Mucosa↗
PubMed2001

The AML1/MTG8 Fusion Transcript in t(8;21) Positive AML and its Implication for the Detection of Minimal Residual Disease; Malignancy.

The reciprocal translocation t(8;21)(q22;q22) is one of the most frequent chromosomal aberrations in acute myeloblastic leukemia (AML). At the molecular level, this aberration rearranges the gene for the AML1-transcription factor on chromosome 21, which is essential for normal hematopoiesis, to the MTG8 gene on chromosome 8, thereby leading to a specific AML1/MTG8 fusion mRNA. This fusion gene is involved in leukemogenesis presumably by interfering with normal AML1-dependent transcriptional regulation. AML patients with t(8;21) have a favourable response to chemotherapy and a relatively good prognosis after intensive consolidation treatment with high dose AraC or autologous stem cell transplantation. RT-PCR for the specific AML1/MTG8 fusion transcripts can be used for the sensitive detection of residual leukemic cells during and after therapy. However, since a considerable proportion of these patients shows a positive PCR result even in long-term complete hematological remission, the prognostic value of qualitative PCR methods is doubtful. In contrast, quantitative PCR methods might be able to identify patients with a high risk of relapse by serial quantification of minimal residual disease (MRD). Because of its high degree of standardisation and automation, the recently developed real time PCR method can be used for the valid and reproducible detection of MRD in large prospective trials. This technology offers the potential to define the antileukemic efficiency of different treatment elements and the prognostic value of MRD in patients with t(8;21) positive AML.

Journal Article↗
PubMedDate not supplied

Possible role of DNA hypomethylation in the induction of SLE: relationship to the transcription of human endogenous retroviruses.

OBJECTIVE: We investigated the contribution of DNA methyltransferase activity to the transcription of human endogenous retroviruses (HERV), which have been reported to be a plausible causative agent for systemic lupus erythematosus (SLE). METHODS: The reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time quantitative-PCR (RQ-PCR) were used. RESULTS: Our results indicated that treatment with 5-aza-deoxycytidine (5-aza C), a demethylating agent, increased the transcription of messenger RNA (mRNA) for HERV clone 4-1 and decreased mRNA for DNA methyltransferase-1 (DNMT-1; methylation-regulating enzyme) in peripheral blood mononuclear cells (PBMC) from normal individuals. Also, transcription of DNMT-1 mRNA in PBMC from patients with SLE was lower than in cells from normal controls. CONCLUSION: DNA hypomethylation seems to play a significant role in the transcription of HERV clone 4-1 and may be related to the pathogenesis of SLE.

Adult↗
PubMed2005

[Regulating effects of transforming growth factor beta on gene expression of matrix metalloproteinase 13 mRNA in human hyaline chondrocytes].

OBJECTIVE: To investigate the role of transforming growth factor beta (TGF-beta)in the regulation of the gene expression of matrix metalloproteinase 13 (MMP-13) in the human hyaline chondrocytes. METHODS: The human hyaline chondrocytes harvested enzymatically and cultured in DMEM supplemented with 20% fetus calf serum were divided into 7 groups. Group 1 was used as a control, and 1 ng/ml TGF-beta (group 2), 10 ng/ml TGF-beta (group 3), 100 ng/ml TGF-beta (group 4), 1 ng/ml TGF-beta+ 10 ng/ml IL-1beta (group 5), 10 ng/ml TGF-beta+ 10 ng/ml IL-1beta (group 6), and 100 ng/ml TGF-beta+ 10 ng/ml IL-1beta (group 7) were given for 12-hour coculture. The MMP-13 mRNA levels of passaged human hyaline chondrocytes were assessed by reverse transcription-polymerase chain reaction (RT-PCR) and real-time fluorescent quantitative PCR. RESULTS: TGF-beta can increase the MMP-13 mRNA level respectively in the passaged hyaline chondrocytes. In the multi-factor treated groups, TGF-beta can decrease the MMP-13 mRNA level respectively and there was significant difference between groups (P< 0.05). The level of MMP-13 mRNA expression had significant coherence with the dosage of TGF-beta. CONCLUSION: The above results show that human chondrocytes express MMP-13 mRNA. TGF-beta could cause a dose-dependent stimulation on MMP-13 gene expression in human chondrocytes and have a potent effect of antagonizing IL-1beta in osteoarthritis. TGF-beta may play a crucial role in the occurrence and development of osteoarthritis through regulating MMP-13.

Cells, Cultured↗
PubMed2005

[HOX11L2 expression and its clinical significance in paraffin-embedded T-lymphoblastic lymphoma].

OBJECTIVE: To determine the feasibility of detecting target gene expression and evaluate the expression of homeobox gene HOX11L2 as well as its clinical significance in paraffin-embedded T-lymphoblastic lymphoma T-LBL. METHODS: HOX11L2 mRNA was analyzed by RT-PCR and real-time fluorescent quantitative PCR (RQ-PCR) on paraffin-embedded tissues in 54 cases of T-LBL. RESULTS: HOX11L2 expression was observed in 7 cases (13.0%). The age ranged from 5 to 15 years, with a median of 9 years. All of the HOX11L2 expressed patients had prior mediastinal masses, and 5 of them expressed high level of Ki67 antigen (PI> or =80%). Symptoms of CNS invasion were diagnosed in 2 cases. Significant differences in the age ratio (P < 0.05), the level of Ki67 antigen (P < 0.05) and the incidence of mediastinal involvement (P < 0.05) were observed between patients positive or negative for HOX11L2. Patients positive for HOX11L2 had a worse outcome than those negative for HOX11L2, with median survival of 7 and 11.5 month, respectively. No statistic difference was found in sex, clinical stages, CNS and bone marrow involvement between HOX11L2 positive and negative groups. CONCLUSIONS: Detection of target gene expression in paraffin-embedded tissues is feasible by RT-PCR or RQ-PCR. HOX11L2 expression was limited in childhood with T-LBL and probably associated with the development and poor prognosis of some pediatric T-LBL patients.

Feasibility Studies↗
PubMed1996

A novel method for real time quantitative RT-PCR.

A novel approach to quantitative reverse transcriptase polymerase chain reaction (QC RT-PCR) using real time detection and the 5' nuclease assay has been developed. Cystic fibrosis transmembrane transductance regulator (CFTR) target mRNA is reverse transcribed, amplified, detected, and quantitated in real time. A fluorogenic probe was designed to detect the CFTR amplicon. Relative increase in 6-carboxy-fluorescein reporter fluorescent emission is monitored during PCR amplification using an analytical thermal cycler. An internal control template containing the same primer sequences as the CFTR amplicon, but a different internal sequence, has been designed as a control. An internal control probe with a reporter fluorescent dye tetrachloro-6-carboxy-fluorescein was designed to hybridize to the internal control amplicon. The internal control template is placed in each reaction tube and is used for quantitative analysis of the CFTR mRNA. This method provides a convenient and high-throughput format for QC RT-PCR.

Cystic Fibrosis Transmembrane Conductance Regulato↗
PubMed2002

Rapid and sensitive detection of physical status of human papillomavirus type 16 DNA by quantitative real-time PCR.

A rapid quantitative real-time PCR method was employed to quantify the copy number of E2 and E6 genes for analysis of the physical status of human papillomavirus type 16 (HPV-16) DNA. Significant differences with respect to both copy numbers were found when more than 40% of HPV-16 DNA was integrated with disruption of the E2 gene in an experimental model. The physical status of HPV-16 DNA in 50 clinical samples was exclusively episomal in 21 cases (42%), concomitant in 14 cases (28%), and integrated in 15 cases (30%). The prevalence of integrated and/or concomitant forms of HPV-16 DNA increased with progression of cervical disease. Four of 11 cervical intraepithelial neoplasia involved integrated forms of HPV-16 DNA partially or exclusively. This rapid, sensitive technique is useful in the analysis of the physical status of HPV DNA.

Blotting, Southern↗