Yuzhou Wang, Marianne D. Sadar, and Richard Sobel

Molecular Analysis and Characterization of PrEC, Commercially Available Prostate Epithelial Cells

Scientists at the BC Cancer Agency are interested in understanding the molecular events that result in the progression of prostate cancer from an androgen-dependent to androgen-independent stage. At the present time, the lack of appropriate in vitro models for research of the development and progression of cancer continues to be a challenge. While there are in vivo model systems that can faithfully mimic some aspects of prostate cancer, they have limitations so researchers often turn to in vitro cell culture systems because the cells are easily maintained, grow quickly and can be readily manipulated to accommodate a variety of experiments. One of their drawbacks is that the established cell lines are derived from malignant or transformed cells of the cancer. Our research investigating prostate carcinogenesis necessitates that we compare malignant cells and “normal” benign cells. Fortunately, primary cultures of benign cells such as PrECs (prostate epithelial cells) are commercially available and have been used by several groups for investigating the progression of prostate cancer. Since androgens play an important role in prostate cancer by regulating the expression of a number of genes, we grew PrECs in the presence and absence of androgens and tested them for the expression of both lineage- and developmental-specific markers and androgen-regulated genes, including PSA (an important clinical diagnostic marker). We found that the gene expression pattern of PrEC cells was consistent with that reported for pubertal prostate transition epithelial cells, but not adult luminal cells. Therefore we concluded that, depending upon the batch of PrECs, these cells would be an excellent source for experiments investigating pre-adult gene and protein expression patterns but are inappropriate cells to use as “normal” benign luminal epithelial cells prior to adenocarcinoma of the prostate, a disease affecting 13% of men with a median age of diagnosis of 72 years. Richard Sobel, Yuzhou Wang, and Marianne D. Sadar. Molecular Analysis and Characterization of PrEC, Commercially Available Prostate Epithelial Cells, In Vitro Cellular and Developmental Biology – Animal, 42: 34-40, 2006.

Daisuke Eguchi

Kikuo Iwabuchi

A New Cell Line from the Wax Moth Galleria mellonella Linne (Lepidoptera:Pyralididae)

Insect cell lines have become increasingly important in studies on physiology and developmental biology as well as the production of recombinant proteins by the use of the baculovirus expression vector. The wax moth Galleria mellonella is often a serious world-wide pest of beehives, tunneling through the combs feeding on wax and honey. Because of its atypical habitat in beehives characterized by very stable thermal conditions, G. mellonella seems to be a very convenient species for studying temperature-mediated changes in insect physiology. A continuous cell line (GaMe-LF1) has been established from larval fat bodies of G. mellonella with MGM-450 medium. The cells grew at temperatures ranging from 15 to 35 C and the maximum growth rate and the highest cell density of 5.8 x 106 cells/ml was achieved at 25 C. Growth of rearing G. mellonella larvae is extremely delayed at a low temperature (15 C). In contrast, GaMe-LF1 cells normally grew at this temperature, suggesting that the cells had developed a tolerance to low temperature (15 C). The GaMe-LF1 cells were sensitive to 20-hydroxyecdysone, the steroid molting hormone of insects, and most cells produced filamentous cytoplasmic extensions. This cell line may be applicable to studies of insect physiology of thermotolerance and hormonal roles. Daisuke Eguchi and Kikuo Iwabuchi. A New Cell Line from the Wax Moth Galleria mellonella Linne (Lepidoptera:Pyralididae), In Vitro Cellular and Developmental Biology – Animal,42:1-3, 2006.

Huanmin Zhou

Heterogenous Nuclear Transfer Embryos Reconstructed by Bovine Oocytes and Camel (Camelus bactrianus) Skin Fibroblasts and Their Subsequent Development

Production of cloned animals by somatic cell nuclear transfer has been successfully achieved in many mammalian species. The generation of viable cloned offsprings from different somatic cells demonstrated that terminally differentiated mammalian somatic cells could be dedifferentiated and recovered totipotency within enucleated oocytes. Therefore, somatic cell nuclear transfer technology has potential for practical application in the in vitro production of embryos, animal improvement and for the preservation of endangered species. The camel population has been reduced rapidly due to the substitution of agricultural machinery in the last two decades in China. Since there is limited availability of camel oocytes for in vitro production but an abundant supply of bovine oocytes, the use of bovine oocytes as recipient cytoplasm would be an alternative approach for camel somatic cell nuclear transfer research. Consequently, We attempted to reconstruct heterogeneous embryos using camel skin fibroblast cells as the donor karyoplasts and enucleated bovine oocytes as the recipient cytoplasts for examining the reprogramming of camel somatic cell nuclei in enucleated bovine oocyte cytoplasm and the developmental potential of the xenonuclear transfer embryos, and determining the suitability of bovine oocytes as host cytoplasts for camel fibroblast cells. Serum-starved skin fibroblast cells, obtained from adult camel, were electrically fused into enucleated bovine MII oocytes that were matured in vitro. The fused eggs were activated by Inomycin with 2 mM/ml 6-dimethylaminopurine. The activated reconstructed embryos were co-cultured with bovine cumulus cells in synthetic oviduct fluid supplemented with amino acid and 10% fetal calf serum for 168 h. Results showed that 53% of the injected oocytes were successfully fused, 34% of the fused eggs underwent the first egg cleavage, and 100% of them developed to 4- or 16-cell embryo stages. The first completed cleavage of xenonuclear transfer camel embryos occurred between 22-48 h following activation. The study demonstrated that the reconstructed embryos underwent the first embryonic division and that the reprogramming of camel fibroblast nuclei can be initiated in enucleated bovine MII oocytes.Huanmin Zhouand Zhenhua Guo. Heterogenous Nuclear Transfer Embryos Reconstructed by Bovine Oocytes and Camel (Camelus bactrianus) Skin Fibroblasts and Their Subsequent Development. In Vitro Cellular and Developmental Biology – Animal, 42:16-19, 2006.

M. A. Couceiro

F. Afreen

S. M. A. Zobayed

T. Kozai

Enhanced Growth and Quality of St. John’s Wort (Hypericum perforatum L.) Under Photoautotrophic In Vitro Conditions

The major limitations of commercial micropropagation for St. John’s wort (Hypericum perforatum L.) might be related to the use of a photomixotrophic micropropagation system (one that uses sugar-containing medium) during the elongation/rooting stage. The culture conditions of this system are thought to cause poor physiological and morphological development, a low net photosynthetic rate and a high percentage of microbial contamination. On the other hand, the use of a photoautotrophic micropropagation system (one that used sugar-free medium) has showed several advantages over the photomixotrophic system. In the photoautotrophic system, growth and production of high quality transplants can be promoted by optimizing environmental factors. Moreover, a smooth transition to the ex vitro environment with rapid growth after transplanting can be obtained. In the current study, leafy nodal cuttings were cultured under photoautotrophic conditions and the growth and quality were compared with those cultured under photomixotrophic conditions. After 21 d of culture, photoautotrophic conditions enhanced the growth and quality of St. John’s wort plantlets in vitro, and these plantlets showed faster growth after transplanting ex vitro compared with those cultured under photomixotrophic conditions. M. A. Couceiro, F. Afreen, S. M. A. Zobayed, and T. Kozai.Enhanced Growth and Quality of St. John’s Wort (Hypericum perforatum L.) Under Photoautotrophic In Vitro Conditions, In Vitro Cellular and Developmental Biology – Plant, 42: issue 3, pages to be determined.