Seeing the gene therapy: application of gene gun technique to transfect and decolour pigmented rat skin with human agouti signalling protein cDNA.

We developed a gene gun method for the transfer of human agouti signalling protein (ASP) cDNA to alter rat skin colour in vivo. Human ASP cDNA was cloned into a modified cytomegalovirus plasmid and delivered to the skin of Long-Evans rats by gene gun bombardment. Skin pigmentation, body weight and blood sugar of ASP cDNA-transfected rats were recorded against the control group, which were injected with plasmids encoding for green fluorescent protein. The treated skin showed lighter skin colour after 3 days of ASP gene transfection. This depigmentation effect was most prominent on day 14 and the skin gradually returned to its original pigmentation by day 28. Successful transfection of ASP gene in skin and hair follicles, as well as downregulation of melanocortin-1 receptor (MC1R) and tyrosinase expression upon treatment, was confirmed using immunohistochemistry and Western blot analysis. Body weight and blood sugar in the treated rats did not show statistically significant differences as compared to control groups. These observations demonstrate that gene transfer using the gene gun method can induce high cutaneous ASP production and facilitate a switch from dark to fair colour without systemic pleiotropic effects. Such a colour switch may be that ASP is acting in a paracrine fashion. In addition, this study verifies that ASP exerts its functions by acting as an independent ligand that downregulates the melanocyte MC1R and tyrosinase protein in an in vivo system. Our result offers new, interesting insights about the effect of ASP on pigmentation, providing a novel approach to study the molecular mechanisms underlying skin melanogenesis.

Suppression of proline-directed protein kinase F(A) potentiates apoptotic induction and greatly enhances chemosensitivity in human acute lymphoblastic leukemia cells.

BACKGROUND: Previously, the authors reported that specific antisense suppression of overexpressed proline-directed protein kinase (PDPK) F(A) enhances the chemosensitivity of various clinical anticancer drugs up to > 100-fold in human prostate carcinoma cells, suggesting an association of PDPK F(A) with drug resistance in human malignancies. METHODS: In this report, by using a similar approach, the authors demonstrate further that the suppression of PDPK F(A) enhances even more dramatically the chemosensitivity of clinically used anticancer drugs in various types of human acute lymphoblastic leukemia (ALL) cells. RESULTS: Compared with parental and control transfected cells, transduced ALL cells (both Jurkat and CCRF-CEM cells) with low levels of PDPK F(A) displayed an enhanced sensitivity to vincristine, vinblastine, paclitaxel, methotrexate, doxorubicin, and daunorubicin. Estimation of the 50% inhibitory concentration (IC(50)) index further revealed that the transduced cells displayed up to > 3000-fold drug sensitivity, and there was a correlation between suppressed levels of PDPK F(A) and drug sensitivity. A mechanistic study further revealed that the enhanced chemosensitivity in transduced ALL cells was due mainly to the potentiation of apoptotic induction. CONCLUSIONS: Taken together, the results demonstrate that the suppression of overexpressed PDPK F(A) greatly enhances the chemosensitivity of various clinical anticancer drugs in both types of human ALL cells, providing initial evidence for an important role of this PDPK in controlling multidrug resistance of ALL.

Suppression of proline-directed protein kinase F(A) expression inhibits the growth of human chronic myeloid leukaemia cells.

Initial studies revealed that proline-directed protein kinase F(A) (PDPK F(A)) was overexpressed in various cancerous tissues relative to normal controls. However, the functional role of overexpressed PDPK F(A) in cancer remains to be established. In this report, we explore the potential role of PDPK F(A) in leukaemia cell growth by investigating the effects of partial inhibition of this kinase on the malignant phenotype of human chronic myeloid leukaemia cells (K562). Cloning of PDPK F(A) cDNA and its recombinant antisense expression vector and PDPK F(A)-specific antibody were successfully developed. Two stable antisense clones of K562 cells were subcloned which expressed 70% and 45% of PDPK F(A) respectively, compared with control-transfected clone in both immunoprecipitate activity assay and immunoblot analysis. In sharp contrast, these two antisense clones expressed no significant suppression of any other related PDPK family members, indicating the specificity of these two antisense clones. Moreover, these antisense clones proportionally and potentially exhibited cell growth retardation, poor clonogenic growth in soft agar and loss of serum independence. The results demonstrate that specific antisense suppression of PDPK F(A) is sufficient to interfere with the growth of K562 cells, indicating that PDPK F(A) is essential for human chronic myeloid leukaemia cell growth.