A mammalian expression plasmid pcDNA3. 1-hCC10 was constructed and identified, then CC10 protein expression in A549 lung cancer cell line was detected. A 273 bp cDNA fragment was amplified from the total RNA of normal lung tissue by using RT-PCR and cloned into expression plasmid cDNA3. 1, and the recombinant plasmid was identified by employing double digestion restriction enzymes Hind III and BamH I and the cDNA sequence was assayed by the Sanger dideoxy-mediated chain termination method. The segment was then transfected into the A549 lung cancer cell line. The protein expression of CC10 was detected by immunofluorescence and Western blot. Our results showed that the cDNA fragment included the entire coding region (273 bp). The recombinant eukaryotic cell expression vector of pcDNA3. 1-hCC10 was successfully constructed, and the sequence of the insert was identical to the published sequence. A549 cells line transfected with the pcDNA3. 1-hCC10 expressed high level of CC10 protein. The recombinant plasmid cDNA3. 1-hCC10 may serve as an effective tool for the study of tumorogenesis and tumor treatment.
Adenocarcinoma/*metabolism ; Adenocarcinoma/pathology ; Cell Line, Tumor ; Genetic Vectors ; Lung Neoplasms/*metabolism ; Lung Neoplasms/pathology ; Recombinant Proteins/biosynthesis ; Recombinant Proteins/genetics ; Transfection ; Uteroglobin/biosynthesis ; Uteroglobin/*genetics

OBJECTIVETo investigate the expression of the human mammoglobin (hMAM) mRNA in bone marrow and its clinical significance in the breast cancer patient.

METHODSExpression of hMAM mRNA was detected using nested reverse transcription polymerase chain reaction (RT-PCR) in the bone marrow aspiration sample from 75 breast cancer patients, 15 patients with benign breast lesions and 8 healthy volunteers as control. The possible correlation of hMAM mRNA expression with clinico-pathological parameters and related molecular markers such as Ki67, p53 and VEGF were analyzed.

RESULTSThe sensitivity of RT-PCR in this series reached 10(-6). The hMAM mRNA was found to be positively expressed by RT-PCR in 21 of 75 breast cancer patients with a positive rate of 28.0%. However, hMAM mRNA expression was not detected in the bone marrow aspiration samples from patients with benign breast lesions and healthy volunteers. The hMAM mRNA expression was positively correlated with axillary nodal involvement and progesterone receptor (PR) status (P < 0.05) as well as Ki67 expression in breast cancer tissue (chi2 = 4.936, P = 0.026), but not with age, tumor size, clinical stage, or estrogen receptor (ER) status (P > 0.05).

CONCLUSIONRT-PCR is quite sensitive and has a high specificity in detecting the presence of hMAM mRNA in the bone marrow from breast cancer patients. Thereupon, hMAM mRNA may be useful as a molecular biomarker in detecting disseminated tumor cells (DTC) in the bone marrow of breast cancer patients. Positive hMAM mRNA expression result may have an impact upon therapeutic recommendations and patients' prognostic judgement.

Adult ; Aged ; Biomarkers, Tumor ; genetics ; Bone Marrow ; metabolism ; pathology ; Breast ; metabolism ; pathology ; Breast Neoplasms ; genetics ; pathology ; Breast Neoplasms, Male ; genetics ; pathology ; Carcinoma, Ductal, Breast ; genetics ; pathology ; Female ; Fibroadenoma ; genetics ; pathology ; Humans ; Ki-67 Antigen ; genetics ; Lymphatic Metastasis ; Male ; Mammaglobin A ; Middle Aged ; Neoplasm Proteins ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, Progesterone ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Uteroglobin ; genetics