Postmitotic cells fated to become rod photoreceptors can be respecified by CNTF treatment of the retina.

Lineage analyses of vertebrate retinae have led to the suggestions that cell fate decisions are made during or after the terminal cell division and that extrinsic factors can influence fate choices. The evidence for a role of extrinsic factors is strongest for development of rodent rod photoreceptors ('rods'). In an effort to identify molecules that may regulate rod development, a number of known factors were assayed in vitro. Ciliary neurotrophic factor (CNTF) was found to have a range of effects on retinal cells. Addition of CNTF to postnatal rat retinal explants resulted in a dramatic reduction in the number of differentiating rods. Conversly, the number of cells expressing markers of bipolar cell differentiation was increased to a level not normally seen in vivo or in vitro. In addition, a small increase in the percentage of cells expressing either a marker of amacrine cells or a marker of Muller glia was noted. It was determined that many of the cells that would normally differentiate into rods were the cells that differentiated as bipolar cells in the presence of CNTF. Prospective rod photoreceptors could make this change even when they were postmitotic, indicating that at least a subset of cells fated to be rods were not committed to this fate at the time they were born. These findings highlight the distinction between cell fate and commitment. Resistance to the effect of CNTF on rod differentiation occurred at about the time that a cell began to express opsin. The time of commitment to terminal rod differentiation may thus coincide with the initiation of opsin expression. In agreement with the hypothesis that CNTF plays a role in rod differentiation in vivo, a greater percentage of cells were observed differentiating as rod photoreceptors in mouse retinal explants lacking a functional CNTF receptor, relative to wild-type littermates.

Gene therapy of malignant brain tumors: a rat glioma line bearing the herpes simplex virus type 1-thymidine kinase gene and wild type retrovirus kills other tumor cells.

Tumor cells infected with a retrovirus vector (VIK) containing the herpes simplex virus thymidine kinase (HSV-TK) gene can be selectively killed by treatment with nucleoside analogues, such as ganciclovir. To mediate delivery of the HSV-TK gene to "recipient" tumor cells, "donor" C6 rat glioma cells infected with the VIK vector (C6VIK) were superinfected with wild type Moloney murine leukemia virus (WT Mo-MLV). These modified donor cells (C6VIKWT) produced both wild type retrovirus and the VIK vector. In culture, C6VIKWT cells were 300-fold more sensitive to the toxicity of ganciclovir than were C6VIK cells, suggesting that the presence of wild type retrovirus contributed to the toxicity. Co-culture of C6VIKWT cells with the C6 subline, C6BAG, sensitized the latter to ganciclovir treatment. Nude mice inoculated subcutaneously with a mixture of C6VIKWT and C6BAG cells showed regression of subsequent tumors when treated with ganciclovir. The observations show that tumor cells modified in culture by infection with a retrovirus bearing the HSV-TK gene and wild type retrovirus are not only sensitive to ganciclovir, but can transfer this sensitivity to neighboring "naive" tumor cells in culture and in vivo.

Selective killing of glioma cells in culture and in vivo by retrovirus transfer of the herpes simplex virus thymidine kinase gene.

The thymidine kinase gene (tk) of herpes simplex type 1 virus (HSV-1) was inserted into a retroviral vector under the transcriptional control of the enhancer-promoter element of the Moloney murine leukemia virus long terminal repeat. Replication-defective viral particles were obtained by transfection of vector DNA into the packaging cell line psi2 and were used to infect C6 rat glioma-derived cell lines in culture. The sensitivity of these cells to the toxic effects of the nucleoside analog ganciclovir was found to be significantly increased by transfer of the HSV-1 tk gene. The difference in sensitivity between infected and uninfected cells defined ganciclovir concentrations that could be used to selectively kill essentially all infected cells while sparing uninfected ones. C6 glioma cells introduced subcutaneously into nude mice were highly tumorigenic. Growth of tumors produced from C6-derived cells bearing the HSV-1 tk gene, but not parental C6 cells, could be inhibited by intraperitoneal administration of ganciclovir. This work demonstrates the effectiveness of the thymidine kinase expressed by the HSV-1 ks gene in sensitizing brain tumor cells to the toxic effects of nucleoside analogs. Retrovirus vectors should thus prove useful in the selective delivery of this killer gene to dividing tumor cells in the nervous system, where most endogenous cells are not dividing.