Construction and Stable Expression of a Truncated Human Receptor Tyrosine Kinase Ror1 (Ror1-ECD).

Expression of receptor tyrosine kinase Ror1 in a wide variety of cancers has emerged as a new era focusing on targeting this receptor in cancer therapy. Our preliminary results indicate the presence of a truncated transcript of Ror1 in tumor cells. The truncated Ror1 encompasses extracellular and transmembrane domains, lacking catalytic kinase domain (Ror1-ECD). As enzyme activity is highly dependent on the catalytic domain, we were wondering how this transcript and its encoded protein could play a possible role in tumorigenesis. To understand the function of this truncated transcript and whether or not the encoded protein translocates to the cell surface, we constructed a mammalian expression vector containing exon 1 to exon 8 of human Ror1 gene as a model system. The encoded protein by this construct covers the entire extracellular and transmembrane domains of Ror1. The Chinese Hamster Ovary Cell line (CHO) was used for transfection. Our results showed that this construct could express Ror1-ECD at protein level and also the protein could effectively translocate to the surface of transfected cells. Such model may suggest that a proportion of Ror1 molecules expressed by tumor cells are not full-length Ror1. This notion may be considered when applying flow cytometry using antibodies against Ror1 for screening of tumor cells in order to avoid any miscalculation in the number of Ror1 molecules expressed by tumor cells. Furthermore, such expression may bring about assumptions on functional roles of Ror1-ECD in tumorigenesis, which requires extensive functional studies.

Profiling and authentication of human cell lines using short tandem repeat (STR) loci: Report from the National Cell Bank of Iran.

Assurance of cell line homogeneity and capability of cell contamination detection are among the most essential steps of cell based research. Due to high discriminatory efficiency, low cost and reliability, analysis of short tandem repeats (STR) has been introduced as a method of choice for human cell line authentication. In the present study 13 Combined DNA Index System (CODIS) based STRs along with the gender determination (Amelogenin) gene were utilized to establish a reproducible approach for the authentication of 100 human cell lines deposited in the National Cell Bank of Iran (NCBI), using the polymerase chain reaction (PCR) method. PCR products were subsequently analyzed by polyacrylamide gel electrophoresis (PAGE) and visualized by silver staining followed by gel documentation and software analysis. STR profiles obtained were compared with those of the American Type Culture Collection (ATCC) and the Japanese Collection of Research Bioresource (JCRB) as STR references. We detected 18.8% cross contamination among the NCBI human cell lines. To our knowledge, this is the first report of authentication of human cell lines using the 13 CODIS core STRs combined with Amelogenin.

Reconstitution of the T-cell repertoire following treatment with alemtuzumab (anti-CD52 monoclonal antibody) in patients with B-cell chronic lymphocytic leukaemia.

In this pilot study, T-cell receptor B-variable (TCR-BV) gene usage in CD4 and CD8 T cells was assessed, by real-time polymerase chain reaction, as well as complementarity-determining region 3 (CDR3)-length polymorphism, before and after therapy in five patients with B-cell chronic lymphocytic leukaemia who received alemtuzumab (anti-CD52 monoclonal antibody) as first-line therapy. A decline in expression of most BV family genes in both CD4 and CD8 T cells was observed after alemtuzumab treatment, which was followed by a gradual increase in most BV families during long-term follow-up. After treatment, CDR3-length polymorphism showed an even more restricted pattern in CD4 T cells compared with pretreatment, with a shift towards a monoclonal/oligoclonal pattern. The clonally restricted pattern was significantly reduced in CD4 (P < 0.01) but not in CD8 T cells. This was followed by a gradual increase in the number of peaks within the CDR3 region of the different TCR-BV families, i.e. a polyclonal repertoire, during long-term follow-up. A restricted CDR3 pattern became even more restricted after treatment, but normalised during unmaintained follow-up. These results indicate that perturbations in the T-cell alterations following alemtuzumab are complex and include not only changes in CD4/CD8 T-cell numbers but also a highly restricted T-cell repertoire especially in CD4 T cells.