Animals ; CHO Cells ; Cricetulus ; Doxycycline ; pharmacology ; Immunohistochemistry ; Ki-67 Antigen ; genetics ; metabolism ; Plasmids ; Quality Control ; Staining and Labeling ; methods ; Transfection

OBJECTIVE: To investigate the expression of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) in multiple myeloma (MM) patients, and analyze the effect of doxycycline (DOX) on the expression of MMP-2 and MMP-9 in MM cells. METHODS: The peripheral blood and bone marrow samples of MM patients were collected, and the patients were divided into three groups: newly diagnosed group, remission group and relapsed/refractory group, while the peripheral blood samples of 34 health people and the bone marrow samples of 17 IDA patients were selected as normal control and control group. The levels of MMP-2 and MMP-9 were detected by ELISA. The protein levels of MMP-2 and MMP-9 in H929 cells treated by different concentrations of DOX were analyzed by Western blot. After H929 cells was treated by Akt inhibitor MK-2206 2HCl in combination with DOX, Western blot was used to detect the levels of MMP-2 and MMP-9. RESULTS: The levels of MMP-2 and MMP-9 in newly diagnosed MM patients were higher than those in control (P<0.05), while for the patients in the remission group were decreased, but still higher than those in control. The levels of MMP-2 and MMP-9 were increased again for the patients in relapsed/refractory group, and showed no significant difference as compared with those in newly diagnosed group. The levels of MMP-2 and MMP-9 could be inhibited by 10 mg/L and 15 mg/L DOX treated by H929 cell. The protein levels of MMP-2 and MMP-9 showed no altered in H929 cells treated by 5 nmol/L MK-2206 2HCl alone. DOX exerted more profound inhibitory effect to MMP-2 and MMP-9 expression in H929 cells when Akt inhibitor MK-2206 2HCl was combined with DOX. CONCLUSION The levels of MMP-2 and MMP-9 are increased in MM patients and related to the disease status of MM. DOX can inhibit the expression of MMP-2 and MMP-9 in MM cells, and antagonizing its activation of Akt signaling pathway can further enhance the inhibitory effect.
Doxycycline/pharmacology* ; Humans ; Matrix Metalloproteinase 2/metabolism* ; Matrix Metalloproteinase 9/metabolism* ; Multiple Myeloma/metabolism* ; Proto-Oncogene Proteins c-akt

OBJECTIVE: To investigate the mechanism of the in vitro toxicity of doxycycline to myeloma cell line H929 and also the possible pathway involved its toxicity. METHODS: Myeloma cell line H929 was treated with DOX, MEK inhibitor U0126 or RAS agonist ML-098, either alone or in combination. Then, the expression of p-MEK, caspase-3, caspase-9 and c-Jun in H929 were used to detected by Western blot; the cells proliferation and apoptosis were detected by CCK-8 assay and flow cytometry, respectively. RESULTS: DOX significantly increased the levels of cleaved caspase-3 and caspase-9, and down-regulated the level of p-MEK in H929 (P<0.05). MEK antagonist U0126 significantly increased the levels of cleaved caspase-3 and caspase-9, and down-regulated the level of p-MEK (P<0.05). After Dox combined with ML-098 treatment of H929 cells, the apoptosis rate of H929 cells was lower than that of DOX alone treatment group(P<0.05). Compared with DOX alone treatment group, the expressions of p-MEK and p-ERK1/2 in DOX+ML-098 combined treatment group were increased, and the levels of cleaved caspase-3,9 in H929 cells were decreased (P<0.05). The levels of c-Jun mRNA and protein increased in H929 when treated by DOX alone (P<0.05). CONCLUSION DOX can induce apoptosis of H929 via intrinsic apoptosis pathway, and MEK/ERK pathway and c-Jun possibly play a role in this process.
Apoptosis ; Caspase 3 ; Caspase 9/pharmacology* ; Cell Line, Tumor ; Cell Proliferation ; Doxycycline/pharmacology* ; Humans ; Mitogen-Activated Protein Kinase Kinases/pharmacology* ; Multiple Myeloma

In order to investigate the application of recombinant adeno-associated virus (rAAV) vector containing Tet regulation system and HSVtk gene in cancer gene therapy, pAAV/TRE/HSVtk/Tet-On was constructed and identified with PCR and restriction enzyme digestion. Packaging cells HEK293 were cotransfected with plasmids pAAV/TRE/HSVtk/Tet-On, pAAV-RC and pAAV-helper to produce infectious rAAV, and CsCl2 densitygradient centrifugation method was performed for purification and concentration of rAAV. The viruses were then transduced into MCF-7 cells. The results of dot blot hybridization indicate that the rAAV can transfer the target gene into MCF-7 cells. MTT assay showed that GCV could kill AAV-infected MCF-7 cells under the induction of Dox. The data demonstrated that rAAV containing Tet regulation system and HSVtk gene was successfully obtained, and could be used for further investigation of in vivo and in vitro experiments.
Cell Line, Tumor ; Dependovirus ; genetics ; metabolism ; Doxycycline ; pharmacology ; Ganciclovir ; pharmacology ; Genes, Transgenic, Suicide ; genetics ; Genetic Therapy ; Genetic Vectors ; genetics ; Humans ; Simplexvirus ; enzymology ; genetics ; Thymidine Kinase ; genetics ; Transfection

OBJECTIVEBased on establishment of four rat models of experimental pulmonary hypertension (PH), the authors examined the inhibition of matrix metalloproteinases (MMPs) by doxycycline and its effect on the development of PH and associated pulmonary vascular remodeling.

METHODHealthy male Sprague-Dawley rats (weight 350 g to 400 g) were randomly divided into nine groups: Normal control group (N), four model groups (H, M, P, PM) and their corresponding drug intervention groups (HD, MD, PD, PMD) in which doxycycline was given by gavage at a 20 mg/kg daily dosage. On day 28 (day 35 for PM and PMD models), the animals were catheterized to record mean pulmonary arterial pressure (mPAP) and then sacrificed. Fulton Index [RV/(LV + S)] was measured immediately. Morphometric parameters, including percent vascular wall thickness and muscularization of non-muscularized peripheral pulmonary arterioles were determined microscopically. The activity of MMPs was measured by gelatin zymography in the lung tissue.

RESULTS(1) Rats in all model groups (H, M, P, PM) developed significant pulmonary arterial hypertension and right ventricular hypertrophy in comparison with their corresponding drug intervention groups (HD, MD, PD, PMD) and normal control group (N) (P < 0.01). For example, mPAP (mm Hg)(1 mm Hg = 0.133 kPa):N: 18.10 +/- 1.45, H: 27.20 +/- 1.55, HD: 23.90 +/- 2.13; Fulton Inedx(%):N: 23.41 +/- 1.84, H: 34.44 +/- 2.70, HD: 27.55 +/- 2.45. (2) The percent vascular wall thickness (WT%) and percentage of muscularization of non-muscular pulmonary arterioles were significantly increased in all model groups compared with drug intervention groups and normal group (P < 0.01). For example, WT%:N: 10.90 +/- 3.11, H:41.41 +/- 5.21, HD: 17.73 +/- 3.12; Muscularization(%):N: 13.83 +/- 3.72, H: 44.93 +/- 2.43, HD: 29.89 +/- 4.45. (3) The activity of MMPs was inhibited by doxycycline effectively as assessed by gelatin zymography (P < 0.01). For example, the activity of MMP2 (A x 10(3)):N: 1.43 +/- 0.24, H: 3.58 +/- 0.28, HD: 2.29 +/- 0.31.

CONCLUSIONDoxycycline attenuated PH and associated pulmonary vascular remodeling in all rat PH models. The study suggests that high expression and enhanced activity of MMPs may play a brutial role in the development of PH. Such phenomenon seems to be common in a variety of PH models of different etiology.

Animals ; Disease Models, Animal ; Doxycycline ; pharmacology ; Hypertension, Pulmonary ; metabolism ; physiopathology ; Male ; Matrix Metalloproteinases ; metabolism ; Pulmonary Artery ; metabolism ; Rats ; Rats, Sprague-Dawley

BACKGROUNDRevTet-On gene expression system was used to deliver the suicide gene tk to human breast cancer cell line MCF-7 and control the tk gene expression level. The animal model of human breast cancer on severe combined immune deficiency (SCID) mice was set up to explore the suicide gene therapy by the regulation of Tet-On.

METHODSHerpes simplex virus-thymidine kinase (HSVtk) gene was inserted into the plasmid pRevTRE and the recombinant retroviral vector pRevTRE/HSVtk was constructed. Using modified calcium phosphate co-precipitation method, two transfections, pRevTRE/HSVtk and pRevTet-On were performed for MCF-7 cell line and selected by hygromycin B and G418. MCF-7 cell line that stably expressed Tet-regulated tk gene was established. HSVtk gene expression in the MCF/TRE/tk/Tet-On cell line was under the control of Doxycycline (Dox). Cell viability was also determined by MTT assay, whereas HSVtk gene expression was analyzed by reverse transcription-PCR (RT-PCR).

RESULTSMCF/TRE/tk/Tet-On cell survival rate was decreased from 100% to less than 20% when ganciclovir (GCV) concentration was increased from 0 to 1000 microg/ml at 1 microg/ml of Dox after 72 hours of GCV administration. At 1 microg/ml of GCV concentration, the cell numbers decreased from 7 x 10(4) cells/ml to 2 x 10(4) cells/ml when Dox concentration was increased from 0 to 1500 ng/ml after 72 hours culture. In addition, bystander effects were generated in vitro when 10% - 25% of transduced MCF-7 cells were mixed in untransduced MCF-7 cells. On the other hand, the human breast cancer models in SCID mice were set up. The tk gene was expressed with the regulated character after MCF/TRE/tk/Tet-On cells were implanted into the female SCID mice 7 days after Dox induction followed by intraperitoneally administration of GCV for 23 days. Subcutaneous tumors in SCID mice that were implanted with MCF/TRE/tk/Tet-On cells shrank remarkably after Dox and GCV administration as compared with the control.

CONCLUSIONThe human breast tumor cells (MCF-7) expressing HSVtk gene can be eradicated by administration of GCV and induced with tetracycline or its derivative Dox in vitro and in vivo.

Animals ; Breast Neoplasms ; therapy ; Bystander Effect ; Cell Line, Tumor ; Cell Survival ; Doxycycline ; pharmacology ; Ganciclovir ; pharmacology ; Genes, Transgenic, Suicide ; Genetic Therapy ; methods ; Genetic Vectors ; Herpesviridae ; genetics ; Humans ; Mice ; Mice, SCID ; Retroviridae ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Thymidine Kinase ; genetics ; Transfection

During the infection of host cells, IE2 protein is one of the first and most abundantly expressed products of HCMV genome, which plays an important role in the controlling of cell cycle and apoptosis. But the correlation between expression level and anti-apoptotic activity of IE2 protein is still not clear. In this study, we successfully established a HCMV IE2 protein expression cell line that was controlled by Tet-On system. The effect of IE2 protein on cell apoptosis and the expression of p53 was detected under different condition of induction. Our results showed that the IE2 protein could inhibit cell apoptosis induced by TNF-alpha. Additionally, the anti-apoptotic activity of IE2 protein seemed to be relevant to its expression level. However, we failed to detect any difference of p53 expression between the IE2 protein expression and non-expression cells. These data indicated that the IE2 protein might inhibit cell apoptosis through regulating different signal pathways.
Anti-Bacterial Agents ; pharmacology ; Apoptosis ; drug effects ; genetics ; Doxycycline ; pharmacology ; Gene Expression Regulation ; drug effects ; genetics ; HeLa Cells ; Humans ; Immediate-Early Proteins ; genetics ; metabolism ; Plasmids ; genetics ; Trans-Activators ; genetics ; metabolism ; Tumor Suppressor Protein p53 ; metabolism

OBJECTIVETo provide a sound cell source for further ex-vivo gene therapy for chronic pain, we attempt to develop an immortalized rat astrocyte cell line that expresses enkephalin regulated by doxycycline.

METHODSRetrovirus infection method was employed to develop an immortalized rat astrocyte cell line that could express enkephalin regulated by doxycycline. The hPPE gene expression level of immoralized astroyte cells (IAC)/ hPPE was detected by RT-PCR, indirect immunofluorescence staining and radioimmunoassay.

RESULTSIAC carrying Tet-on system transfected with preproenkephalin gene could secrete enkephalin that was regulated by doxycycline in a dose-dependent manner and hPPE gene activation could be repeated in on-off-on cycles through administration or removal of doxycycline.

CONCLUSIONAn immortalized rat astrocyte cell line that secrete enkephalin under the control of doxycycline is established successfully, which provides a research basis for transgenic cell transplantation for analgesia.

Animals ; Anti-Bacterial Agents ; pharmacology ; Astrocytes ; Cell Line, Transformed ; Chronic Disease ; Doxycycline ; pharmacology ; Enkephalins ; genetics ; metabolism ; Genetic Therapy ; methods ; Genetic Vectors ; Neurotransmitter Agents ; genetics ; metabolism ; Pain Management ; Protein Precursors ; genetics ; metabolism ; Rats ; Retroviridae ; genetics

In order to investigate the susceptibility of mixed infection of Ureaplasma Urealyticum (UU) and Mycoplasma Hominis (MH) to 7 kinds of antimicrobial agents and comparison with that of UU infection in NGU patients, the in vitro susceptibility was determined by using microdilution method. The positive results were analyzed. The results showed that the sequence of susceptibility to 7 kinds of antimicrobial agents for both UU infection group and UU-MH mixed infection group was almost the same from the highest susceptibility to the lowest accordingly: Josamycin, Doxycycline, Minocycline, Sparfloxacin, Roxithromycin, Ofloxacin and Azithromycin. The total drug resistance rate for UU-MH mixed infection group (97.67%) was significantly higher than that for UU infection group (44.67%, P < 0.01). The highest drug resistance rate in UU group and UU-MH mixed infection group was 31.33% (Ofloxacin) and 90.48% (Azithromycin) respectively. UU-MH mixed infection showed an increased drug resistance and changes of drug resistance spectrum.
Adult ; Anti-Bacterial Agents ; pharmacology ; Azithromycin ; pharmacology ; Doxycycline ; pharmacology ; Drug Resistance, Bacterial ; Female ; Humans ; Josamycin ; pharmacology ; Male ; Middle Aged ; Minocycline ; pharmacology ; Mycoplasma Infections ; complications ; microbiology ; Mycoplasma hominis ; drug effects ; isolation & purification ; Ofloxacin ; pharmacology ; Superinfection ; Ureaplasma Infections ; complications ; microbiology ; Ureaplasma urealyticum ; drug effects ; isolation & purification

BACKGROUNDAfter T-cell depleted allogeneic bone marrow transplantation, impaired immune reconstitution is a major cause of morbidity and mortality in the recipient. The purpose of this study was to observe the effects of the gene-engineered bone marrow stromal cell line QXMSC1-IL-2 + IL-3 on the reconstitution of T-cell immunity in allo-BMT mice.

METHODSThe bone marrow stromal cell line QXMSC1 was co-transfected with IL-2 and IL-3 genes using a Tet-on gene expression system. T lymphocyte subset counts per spleen were analyzed by flow cytometry. Lymphocyte proliferation response to ConA was examined to evaluate T-cell function. CDR3 spectratyping techniques were performed to evaluate TCR repertoire diversity at various time points post-transplantation.

RESULTSGene engineered bone marrow stromal cell line QXMSC1-IL-2 + IL-3 could express IL-2 and IL-3 [1,300 ng.day(-1).10(-6) cells and 1100 ng.day(-1).10(-6) cells, respectively] under the control of doxycycline. QXMSC1-IL-2 + IL-3 in combination with allogeneic bone marrow could significantly increase the counts of CD(4)(+) and CD(8)(+) T cell, 1.72 and 1.27-fold respectively at week 3 compared with TCD-BMT group (P < 0.01); make CD(4)(+)/CD(8)(+) ratio return to normal level at week 4; enhance splenocytes mitotic response to ConA (P < 0.01), and accelerate restoration of TCR repertoire diversity in the lethally irradiated mice (P < 0.05).

CONCLUSIONThe gene transduced stromal cell line QXMSC1-IL-2 + IL-3 is able to accelerate T-cell immunity in allo-BMT mice.

Animals ; Bone Marrow Cells ; metabolism ; Bone Marrow Transplantation ; immunology ; Cell Line ; Complementarity Determining Regions ; Doxycycline ; pharmacology ; Female ; Interleukin-2 ; genetics ; Interleukin-3 ; genetics ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Stromal Cells ; metabolism ; T-Lymphocytes ; immunology ; Transfection ; Transplantation, Homologous