Naturally occurring hepatitis B virus reverse transcriptase mutations related to potential antiviral drug resistance and liver disease progression

The annual number of deaths caused by hepatitis B virus(HBV)-related disease, including cirrhosis and hepatocellular carcinoma(HCC), is estimated as 887000. The reported prevalence of HBV reverse transcriptase(RT) mutation prior to treatment is varied and the impact of preexisting mutations on the treatment of na?ve patients remains controversial, and primarily depends on geographic factors, HBV genotypes, HBe Ag serostatus, HBV viral loads, disease progression, intergenotypic recombination and co-infection with HIV. Different sensitivity of detection methodology used could also affect their prevalence results. Several genotype-dependent HBV RT positions that can affect the emergence of drug resistance have also been reported. Eight mutations in RT(rtL80I, rtD134N, rtN139K/T/H, rtY141F, rtM204I/V, rtF221Y, rtI224V, and rtM309K) are significantly associated with HCC progression. HBe Ag-negative status, low viral load, and genotype C infection are significantly related to a higher frequency and prevalence of preexisting RT mutations. Preexisting mutations are most frequently found in the A-B interdomain of RT which overlaps with the HBs Ag "a" determinant region, mutations of which can lead to simultaneous viral immune escape. In conclusion, the presence of baseline RT mutations can affect drug treatment outcomes and disease progression in HBVinfected populations via modulation of viral fitness and host-immune responses.

Testicular expression of survivin and human telomerase reverse transcriptase(hTERT)associated with spermatogenic function in infertile patients

Aim： To characterize the coexpression of survivin, an inhibitor of apoptosis （IAF）, and human telomerase reverse transcriptase （hTERT） in human testes with varying spermatogenic function. Methods： Transcript levels of survivin mRNA and hTERT mRNA were determined in normal testes （n = 11） and testes with defective spermatogenesis （n = 28） using real-time reverse-transcription polymerase chain reaction （RT-PCR）. The histological work-up was performed according to a modified Johnsen score. Results： Expressions of both survivin and hTERT were highest at median levels of 96.8 and 709 in normal spermatogenesis and dropped to 53.3 and 534 in testes with postmeiotic spermatogenic arrest （n = 10）. In severe spermatogenic failure （n = 18）, survivin expression was lacking in most specimens （n = 16）, whereas at least low levels of testicular hTERT expression were largely detectable with a normalized expression of 73 in premeiotic spermatogenic arrest （n = 7） and 45 in patients with Sertoli cell-only syndrome （SCOS） （n = 3）. Both survivin and hTERT expressions increased with a progressing Johnsen score （P for trend = 0.001）. Conclusion： Although both survivin and hTERT are correlated with spermatogenic function, they show different expression patterns in testes of infertile patients. These findings substantiate results from studies in the rodent testis suggesting a predominant expression of survivin in meiotically dividing germ cells. （Asian J Andro12006 Jan; 8： 95-100）

Transplantation of human telomerase reverse transcriptase gene-transfected Schwann cells for repairing spinal cord injury

Transfection of the human telomerase reverse transcriptase（h TERT）gene has been shown to increase cell proliferation and enhance tissue repair.In the present study,h TERT was transfected into rat Schwann cells.A rat model of acute spinal cord injury was established by the modified free-falling method.Retrovirus PLXSN was injected at the site of spinal cord injury as a vector to mediate h TERT gene-transfected Schwann cells（1×10^（10）/L;10μL）or Schwann cells（1×10^（10）/L;10μL）without h TERT gene transfection.Between 1 and 4 weeks after model establishment,motor function of the lower limb improved in the h TERT-transfected group compared with the group with non-transfected Schwann cells.Terminal deoxynucleotidyl transferase-mediated d UTP nick-end labeling and reverse transcription-polymerase chain reaction results revealed that the number of apoptotic cells,and gene expression of aquaporin 4/9 and matrix metalloproteinase 9/2decreased at the site of injury in both groups;however,the effect improved in the h TERT-transfected group compared with the Schwann cells without h TERT transfection group.Hematoxylin and eosin staining,PKH26 fluorescent labeling,and electrophysiological testing demonstrated that compared with the non-transfected group,spinal cord cavity and motor and sensory evoked potential latencies were reduced,while the number of PKH26-positive cells and the motor and sensory evoked potential amplitude increased at the site of injury in the h TERT-transfected group.These findings suggest that transplantation of h TERT gene-transfected Schwann cells repairs the structure and function of the injured spinal cord.

Neuroprotective effects of human telomerase reverse transcriptase on beta-amyloid fragment 25-35-treated human embryonic cortical neurons

BACKGROUND： Numerous current studies have suggested that human telomerase reverse transcriptase （hTERT） gene has neuroprotective effects and can inhibit apoptosis induced by various cytotoxic stresses; however, the mechanism of action remains unknown. OBJECTIVE： To evaluate the neuroprotective effects and possible mechanism of action of hTERT gene transfection in human embryonic cortical neurons treated with beta-amyloid fragment 25-35 （AI325-35）. DESIGN, TIME AND SETTING： The randomized, controlled and molecular biological studies were performed at the Department of Anatomy and Brain Research, Zhongshan School of Medicine, Sun Yat-sen University, China, from September 2005 to June 2008. MATERIALS： AdEasy-1 Expression System was gifted by Professor Guoquan Gao from Sun Yat-Sen University, China. Human cortical neurons were derived from 12-20 week old aborted fetuses, obtained from the Guangzhou Maternal and Child Health Hospital, China. Mouse anti-Odk5 and mouse anti-p16 monoclonal antibodies （Lab Vision, USA）, and mouse anti-hTERT monoclonal antibody （Epitomics, USA）, were used in this study. METHODS： （1） Recombinant adenovirus vectors, encoding hTERT （Ad-hTERT） and green fluorescent protein （Ad-GFP）, were constructed using the AdEasy-1 Expression System. Human embryonic cortical neurons in the Ad-hTERT group were transfected with Ad-hTERT for 1-21 days. Likewise, human embryonic cortical neurons in the Ad-GFP group were transfected with Ad-GFP for 1-21 days. Human embryonic cortical neurons in the control group were cultured as normal. （2） Human embryonic cortical neurons in the Ad-hTERT group were treated with 10 pmol/L Aβ25-35 for 24 hours. Normal human embryonic cortical neurons treated with 10 pmol/Lβ25.35 for 24 hours served as a model group. Human embryonic cortical neurons in the Ad-GFP and control groups were not treated with Aβ25-35. MAIN OUTCOME MEASURES： Expression of hTERT in human embryonic cortical neurons was evaluated by immunocytochemical staining and Western blot assay. Telomerase activity was measured using a PCR-based telomeric repeat amplification protocol （TRAP） ELISA kit. Neural activity in human embryonic cortical neurons was examined by MTT assay; apoptosis was measured using TUNEL assay; and Cdk5 and p16 protein expressions were measured by Western blot. RESULTS： Expression of hTERT protein was significantly increased and peaked at day 3 post-transfection in the Ad-hTERT group. No hTERT expression was detected in the Ad-GFP and control groups. Telomerase activity was significantly greater in the Ad-hTERT group compared with the Ad-GFP and control groups （P 〈 0.01）. Compared with the control group, cell activity was significantly decreased （P 〈 0.05）, and cell apoptotic rate, Cdk5 and p16 expression were significantly increased （P 〈 0.01） in the model group. Compared with the model group, cell activity was increased in the Ad-hTERT group, and peaked at day 3 post-transfection （P 〈 0.05）. Neuroprotective effects also peaked at day 3 post-transfection; and the apoptotic rate, Cdk5 and p16 expression significantly decreased （P 〈 0.01）. CONCLUSION： Expression of hTERT in human embryonic cortical neurons can relieve Aβ25-35-induced neuronal apoptosis. The possible mechanism by which hTERT produces these neuroprotective effects may be associated with inhibition of Cdk5 and p16 expression.

Quantification of human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) mRNA in testicular tissue of infertile patients

Aim: To evaluate the quantitative detection of human telomerase RNA (hTR) and human telomerase reverse tran-scriptase (hTERT) mRNA as diagnostic parameters in the workup of testicular tissue specimens from patients presentingwith non-obstructive azoospermia. Methods: hTR and hTERT mRNA expression were quantified in 38 cryopre-served testicular tissue specimens by fluorescence real-time reverse transcription - polymerase chain reaction (RT-PCR)in a LightCycler(r). This was paralleled by conventional histological workup in all tissue specimens and additionalsemithin sectioning preparation in cases with maturation arrest (n = 12) and Sertoli-cell-only syndrome (n = 12). Re-sults; The average normalized hTERT expression (N_(hTERT)) was 131.9±48.0 copies (mean ± SD) in tissue speci-mens with full spermatogenesis, N_(hTERT) = 51.2 ±17.2 copies in those with maturation arrest and N_(hTERT) = 2.7 ± 2.4copies in those with Sertoli-cell-only syndrome (SCOS). The discriminant analysis showed that detection of N_(hTERT)(N_(hTR)) had a predictive value of 86.8% (55.3%) for correct classification in one of the three histological subgroups.Conclusion; Our results demonstrate that quantitative detection of hTERT mRNA expression in testicular tissue en-ables a molecular-diagnostic classification of gametogenesis. Quantitative detection of hTERT in testicular biopsies isthus well suited for supplementing the histopathological evaluation. (Asian J Androl 2001 Dec; 3: 263 - 270)

Inhibition of telomerase with human telomerase reverse transcriptase antisense increases the sensitivity of tumor necrosis factor-α-induced apoptosis in prostate cancer cells

Aim： To investigate the effect of inhibition of telomerase with human telomerase reverse transcriptase （hTERT） antisense on tumor necrosis factor-α （TNF-α-induced apoptosis in prostate cancer cells （PC3）. Methods： Antisense phosphorothioate oligodeoxynucleotide （AS PS-ODN） was synthesized and purified. Telomerase activity was measured using the telomeric repeat amplification protocol （TRAP） and polymerase chain reaction enzyme-linked immunoassay （PCR-ELISA）. hTERT mRNA was measured by reverse transcription PCR （RT-PCR） assay and gel-image system, hTERT protein was detected by immunochemistry and flow cytometry. Cell viability was detected by 3-（4, 5-dimethylthiazol-2-yl）-2,5-Diphenyltetrazolium （MTT） assay. Cell apoptosis was observed by morphological method and determined by flow cytometry. Results： The telomerase activity decreased with time after hTERT AS PS-ODN treatment. The levels of hTERT mRNA decreased with time after hTERT AS PS-ODN treatment, which appeared before the decline of the telomerase activity. The percentage of positive cells of hTERT protein declined with time after hTERT AS PS-ODN treatment, which appeared after the decline of hTERT mRNA. There was no difference in telomerase activity, hTERT mRNA and protein levels between hTERT sense phosphorothioate oligodeoxynucleotide （S PS-ODN） and the control group. The cell viability decreased with time after hTERT AS PS-ODN combined with TNF-α treatment. The percentage of apoptosis increased with time after hTERT AS PS-ODN combined with TNF-α treatment. There was no difference in cell viability and the percentage of apoptosis between hTERT S PS-ODN and the control group. Conclusion： hTERT AS PS-ODN can significantly inhibit telomerase activity by downregulating the hTERT mRNA and protein expression, and inhibition of telomerase with hTERT antisense can enhance TNF-α- induced apoptosis of PC3 cells.

Telomerase Activity and Telomerase Reverse Transcriptase Expression Induced by Selenium in Rat Hepatocytes

Objective To investigate the effects of sodium selenite on telomerase activity, apoptosis and expression of TERT, c-myc and p53 in rat hepatocytes. Methods Selenium at doses of 2.5, 5.0, and 10 μmol/kg was given to SD rats by garage. In rat hepatocytes, telomerase activity was measured by the telomeric repeat amplification protocol （TRAP）, apoptosis was detected by flow cytometry, and expressions of telomerase reverse transcriptase （TERT）, c-myc and p53 were analyzed by reverse transcription-polymerase chain reaction （RT-PCR）. c-Myc and P53 proteins were detected by immunochemistry. Results Selenium at doses of 2.5, 5.0, and 10 μmol/kg significantly increased hepatocellular telomerase activity and induced apoptosis in a dose-dependent manner. Although selenium at doses of 2.5, 5.0, and 10 μmol/kg displayed no obvious enhancing effect on the TERT mRNA expression in rat hepatocytes （P〉0.05）, it significantly increased the c-myc mRNA and p53 mRNA expression at the dose of 10 μmol/kg （P〈0.05）. Selenium at doses of 5.0 and 10 μmol/kg obviously increased the content of P53 protein in rat hepatocytes, but only at the dose of 10 μmol/kg, it significantly promoted the value of c-Myc protein in them. Conclusion Selenium can slightly increase telomerase activity and TERT expression, and significantly induce apoptosis and over-expression of c-myc and p53 at relatively high doses. The beneficial effects of selenium on senescence and aging may be mediated by telomerase activation and expression of TERT, c-myc, and p53 in rat hepatocytes.

Unique case of oligoastrocytoma with recurrence and grade progression:Exhibiting differential expression of high mobility group-A1 and human telomerase reverse transcriptase

Mixed gliomas, primarily oligoastrocytomas, account for about 5%-10% of all gliomas. Distinguishing oligoastrocytoma based on histological features alone has limitations in predicting the exact biological behavior, necessitating ancillary markers for greater specificity. In this case report, human telomerase reverse transcriptase(hT ERT) and high mobility group-A1(HMGA1); markers of proliferation and stemness, have been quantitatively analyzed in formalin-fixed paraffin-embedded tissue samples of a 34 years old patient with oligoastrocytoma. Customized florescence-based immunohistochemistry protocol with enhanced sensitivity and specificity is used in the study. The patient presented with a history of generalized seizures and his magnetic resonance imaging scans revealed infiltrative ill-defined mass lesion with calcified foci within the left frontal white matter, suggestive of glioma. He was surgically treated at our center for four consecutive clinical events. Histopathologically, the tumor was identified as oligoastrocytoma-grade Ⅱ followed by two recurrence events and final progression to grade Ⅲ. Overall survival of the patient without adjuvant therapy was more than 9 years. Glial fibrillary acidic protein, p53, Ki-67, nuclear atypia index, pre-operative neutrophillymphocyte ratio, are the other parameters assessed. Findings suggest that hT ERT and HMGA1 are linked to tumor recurrence and progression. Established markers can assist in defining precise histopathological grade in conjuction with conventional markers in clinical setup.

Naturally occurring mutations in the reverse transcriptase region of hepatitis B virus polymerase from treatment-na?ve Korean patients infected with genotype C2

AIM To report naturally occurring mutations in the reverse transcriptase region(RT) of hepatitis B virus(HBV) polymerase from treatment na?ve Korean chronic patients infected with genotype C2.METHODS Here, full-length HBV reverse transcriptase RT sequences were amplified and sequenced from 131 treatment na?ve Korean patients chronically infected with hepatitis B genotype C2. The patients had two distinct clinical statuses: 59 patients with chronic hepatitis(CH) and 72 patients with hepatocellular carcinoma(HCC). The deduced amino acids(AAs) at42 previously reported potential nucleos(t)ide analog resistance(NAr) mutation positions in the RT region were analyzed. RESULTS Potential NAr mutations involving 24 positions were found in 79 of the 131 patients(60.3%). Notably, AA substitutions at 2 positions(rt184 and rt204) involved in primary drug resistance and at 2 positions(rt80 and rt180) that functioned as secondary/compensatory mutations were detected in 10 patients(1 CH patient and 9 HCC patients) and 7 patients(1 CH and 6 HCC patients), respectively. The overall mutation frequencies in the HCC patients(3.17%, 96/3024 mutations) were significantly higher than the frequencies in the CH patients(2.09%, 52/2478 mutations)(P = 0.003). In addition, a total of 3 NAr positions, rt80, rt139 and rt204 were found to be significantly related to HCC from treatment na?ve Korean patients. CONCLUSION Our data showed that naturally occurring NAr mutations in South Korea might contribute to liver disease progression(particularly HCC generation) in chronic patients with genotype C2 infections.

Identification and Characterization of Reverse Transcriptase Fragments of Long Interspersed Nuclear Elements (LINEs) in the <i>Morus notabilis</i>Genome

Reverse transcriptase (rt) fragments from LINE retrotransposons in the mulberry genome were analyzed in terms of heterogeneity, phylogeny, and chromosomal distribution. We amplified and characterized conserved domains of the rt using degenerate primer pairs. Sequence analyses indicated that the rt fragments were highly heterogeneous and rich in A/T bases. The sequence identity ranged from 31.8% to 99.4%. Based on sequence similarities, the rt fragments were categorized into eight groups. Furthermore, similar stop codon distribution patterns among a series of clones in the same group indicated that they underwent a similar evolutionary process. Interestingly, phylogenetic analyses of the rt fragments isolated from mulberry and 13 other plant species revealed that two distantly related taxa (mulberry and Paeonia suffruticosa) grouped together. It does not appear that this phenomenon resulted from horizontal transposable element transfer. Fluorescence in situ hybridization analysis revealed that most of the rt fragments were concentrated in the subtelomeric and pericentromeric regions of the mulberry chromosomes, but that these elements were not abundant in the mulberry genome. Future studies will focus on the potential roles of these elements in the subtelomeric and pericentromeric regions of the mulberry genome.

Molecular Modeling of Quinoline-Based Compounds as Potential Dual Inhibitors of Reverse Transcriptase and Integrase of HIV

As follow-up of our past publication?[1], we propose that quinolones (as part of the pyridinone family) are capable to increase the number of interactions with HIV reverse transcriptase (RT) or integrase (IN) by adding a halogen in position C-8 of aromatic portion of the quinolones. This addition could help with the activity of dual inhibitors of RT and IN. In this work, we add a chlorine atom with the rationale to identify in the docking simulations a halogen interaction with the oxygen in the near aminoacids in the binding pockets of RT and IN enzymes. Our docking studies started with RT and 320 structures. Later, we took 73 structures with good results in docking with RT. The structures that we choose contain ester or acids groups in C-3 due the structural similarity with groups in charge to interact with the Mg++ ions in Elvitegravir. In conclusion, we obtained 14 structures that could occupy the allosteric pocket of RT and could inhibit the catalytic activity of IN, for this reason could be dual inhibitors. A major perspective of this work is the synthesis and testing of the potential dual inhibitors designed.

SYBR Green I-based product-enhanced reverse transcriptase assay for quantification of retroviral PFV and detection of the divalent cation preference of PFV RT

Dear Editor,Prototype foamy virus(PFV)belongs to the genus Spumavirus in the Spumaretrovirinae subfamily of Retroviridae.Although PFV and HIV have much in common,research into PFV has lagged far behind that into HIV,as PFV appeared to be non-pathogenic both in accidentally infected humans and in experimentally infected animals.In recent decades,however,more attention has been focused on PFV because it seems to

Screening of new non-nucleoside reverse transcriptase inhibitors of HIV-1 based on traditional Chinese medicines database

HIV- 1 RT is an important target for the treatment of AIDS. There are two major classes of antiviral agents that inhibit HIV- 1 RT have been identified, nucleoside RT inhibitors （NRTIs） and non-nucleoside RT inhibitors （NNRTIs）. In this report, a noval class of non-nucleoside compound with potential RT inhibitory activity were found from the traditional Chinese medicines database （TCMD） using a combination of virtual screening, docking, molecular dynamic simulations, where results were ranked by scoring function of the docking tool. The result indicates that M4753 （a compound derived from TCMD） has not only the lowest bonding energy but also the best match in geometric conformation with the forthcoming NNRTIs. Accordingly M4753 might possibly become a promising lead compound of NNRTIs for AIDS therapy.

Development of Reverse Transcriptase Loop-Mediated Isothermal Amplification for Rapid and Visualized Detection of Classical Swine Fever Virus

[ Objective] To develop a rapid and visualized detection method of classical swine fever virus （CSFV） using reverse transcriptase loopmediated isothermal amplification （RT-LAMP）. [ Method ] A total of six special primers were designed based on the conserved sequences of CSFV gene. After optimizing, the reaction of RT-LAMP was carded out at 63℃ for 45 rain. The RT-LAMP products were analyzed by agarose gel electro- phoresis. The sensitivity, specificity and repeatability were verified, respectively. [ Result] The RT-LAMP method could be used for detecting CSFV rather than six generic viruses. The sensitivity of RT-LAMP was 100 times higher than that of RT-PCR. The detection of 27 clinical samples by RT- LAMP and RT-PCR showed that RT-LAMP is more reliable and convenient. [ Conclusion] The RT-LAMP method is sensitive and reliable for the detection of CSFV.

Expression of Telomerase Reverse Transcriptase during the Malignant Transformation of Cadmium-Induced Cells

The objective of the present study was to investigate human telomerase reverse transcriptase (hTERT) mRNA and protein expressions during the cadmium chloride-induced malignant transformation of human bronchial epithelial (16HBE) cells. Fluorescence quantitative PCR (FQ-PCR) and Western blot analyses were performed to detect the hTERT mRNA and protein expressions in normal 16HBE cells, cadmium chloride-transformed 16HBE cells, and tumorigenic cells from nude mice inoculated with cadmium chloride-transformed 16HBE cells. Under the inner standard of GAPDH, the hTERT mRNA expression was significantly higher at different stages of malignant transformation (cadmium chloride-transformed 16HBE cells at passages 15 and 35 and tumorigenic cells from nude mice) than in normal 16HBE cells, and increased with the development of malignancy (P < 0.01). In addition, hTERT protein expression increased with the development of malignancy. These findings demonstrate that hTERT expression is related to cadmium chlorideinduced malignant transformation. Cadmium chloride-induced malignant transformation is involved in changes in the hTERT activity, and might be an early event in cadmium chloride-induced malignant transformation.

Covariation of mutation pairs expressed in HIV-1 protease and reverse transcriptase genes subjected to varying treatments

A previous study, focused on the correlation of muta-tion pairs of synonymous (S) and asynonymous (A) mutations, distinguished only between the treated and untreated data of protease and reverse tran-scriptase (RT) of HIV-1 subtype B. It is well known that single mutation patterns in HIV-1 are treat-ment-specific. It logically follows that covariation between mutations will also be treatment specific. Thus, our motivation is to give a more in depth study of the covariation between mutation pairs, analyzing not only treated and untreated, but what specific treatments were used, and how they affected the co-variation between the mutations differently. We in-tended to further deepen this study by analyzing the covariation of mutations in protease and RT in dif-ferent subtypes of HIV-1. We found that virus sam-ples subjected to antiretroviral Protease- and RT- inhibitors do show different patterns of mutation covariation in B-subtype protease and RT of HIV-1, while maintaining the same overall trend. covariation will tend to be higher and more distinct from and covariation after treatment. The same trend continues in protease and RT re-gardless of subtype. We also found the highly cova-ried codon positions, position pairs, and position- covariation clusters in protease, affected by different treatments. Most of them are well known major drug-resistance sites for these treatments.

DETECTION OF BREAST CANCER MICROMETASTASES IN BONE MARROW USING REVERSE-TRANSCRIPTASE CHAINREACTION AND SOUTHERN HYBRIDIZATION

Objective: The aim of this study was to detect micrometastases in bone marrow of primary breast cancer patients, and compare with other clinical parameters. Methods: Cytokeratin 19 (CK-19) gene mRNA expression was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot hybridization. Human breast cancer cell line T47D was mixed with bone marrow cells in different proportions. The positive detection rate was compared among RT-PCR, Southern blotting and immunohistochemistry (IHC) methods. Results: Cytokeratin 19 gene was expressed in all 6 positive control samples while the expression was not seen in 8 negative control samples. In all 54 patients 14 cases were CK-19 positive (25.9%) by RT-PCR, another positive signal was obtained in 5/54 (9.3%) of bone marrow samples by Southern blotting. The total positive cases are 19/54 (35.2%). CK-19 IHC+ cells were detected at a dilution of one T47D cell in 5×104 bone marrow cells, while the sensitivity detected by PCR and Southern blot hybridization was at 1∶5×105 and 1∶1×106, respectively. This demonstrates that RT-PCR and Southern blotting was at least 20 times more sensitive than the IHC method. The micrometastases positive rate of the larger tumor size group (>5.0 cm) was significantly (P<0.05) greater than that of the smaller tumor size group (0–2.0 cm). Conclusion: detection of micrometastases in bone marrow by RT-PCR and Southern blotting, using CK-19 as a biological marker, is highly sensitive and it is a method to be used for anticipating the prognosis of breast cancer patients.

COMBINED DETECTION OF BREAST CANCER MICROMETASTASES IN THE LYMPH NODES AND BONE MARROW USING REVERSETRANSCRIPTASE CHAIN REACTION AND SOUTHERN HYBRIDIZATION

Objective: The presence of lymph nodes and bone marrow micrometastases of patients with breast carcinoma by immunohistochemistry (IHC) methods has been strongly correlated to early recurrence and shorter overall survival. The aim of this study was to detect micrometastases in matched sample pairs of lymph nodes and the bone marrow of primary breast cancer patients using a more sensitive method, and compare with other clinical parameters. Methods: Cytokeratin 19 (CK-19) gene mRNA expression was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot hybridization. Human breast cancer cell line T47D was mixed with bone marrow cells at different proportions. The positive detection rate was compared among RT-PCR, Southern blotting and IHC methods. Results: Cytokeratin 19 gene was expressed in all 6 positive control samples, while the expression wasn’t seen in 18 negative control samples. CK-19 IHC positive cells were detected at a dilution of one T47D cell in 5×105 bone marrow cells, while the sensitivity detected by PCR and Southern blot hybridization was at 1:5×104 and 1:106, respectively. In the samples from the 35 patients, we found CK-19 positive cells in 2 cases (5.7%) by IHC. CK-19 gene expression signal was detected in 14/35 (40%) by RT-PCR, and 17/35 (48.6%) by southern blotting. Four cases were micrometastases positive both in lymph node and bone marrow (11.4%). There was no correlation between CK-19 detection and other clinical parameters. Conclusion: combined detection of micrometastases in lymph node and bone marrow by RT-PCR and Southern blotting, using CK-19 as a biological marker, is a highly sensitive method for breast cancer.