ABSTRACT
'Bystander' killing of adjacent wild-type tumor cells was seen when tumors transduced with the herpes thymidine kinase gene were treated with the antiviral agent ganciclovir (GCV). Some tumors were 'bystander-sensitive' while others were 'bystander-resistant'. Mixtures of different 'sensitive' tumor lines showed cross-transfer of bystander killing, while in mixtures of 'resistant' with 'sensitive' tumors, the resistant phenotype was predominant. Using 3H-GCV with 'sensitive' mixtures, phosphorylated 3H-GCV was found in both herpes thymidine kinase transduced and unmodified cells, while 'resistant' cell combinations showed little or no transfer of phosphorylated GCV between cells. The capacity of intracellularly produced nucleotide toxin to spread from cell to cell within a tumor mass effectively amplifies the apparent efficiency of gene transfer in the tumor and makes feasible the use of this system for therapy of localized cancer.
Subject(s)
Ganciclovir/therapeutic use , Gene Transfer Techniques , Genetic Therapy/methods , Herpes Simplex/enzymology , Thymidine Kinase/genetics , Tumor Cells, Cultured , Adenocarcinoma/therapy , Animals , Antiviral Agents , Biological Transport , Coculture Techniques , Combined Modality Therapy , DNA/biosynthesis , Fibrosarcoma/therapy , Humans , Immunomagnetic Separation , Mice , Mice, Inbred C57BL , PhosphorylationABSTRACT
We have identified a point mutation in one alpha 1(I) collagen allele (COL1A1) of a child with the type IV osteogenesis imperfecta phenotype. When compared to parental and control samples, skin fibroblasts of the proband synthesized two populations of type I collagen molecules. One population was normal; the other was delayed in secretion and electrophoretic migration due to post-translational overmodification. Two-dimensional gel electrophoresis of the CNBr peptides demonstrated a gradient of overmodification beginning near the carboxyl-terminal CB peptides. This predicts that the mutation delaying helix formation is near the carboxyl-terminal end of one of the component chains of type I collagen. The mRNA of the patient was probed with overlapping antisense riboprobes to type I collagen cDNA. Cleavage of a mismatch in RNA/RNA hybrids of RNase A allowed the location of the mutation to a 225-base pair region of alpha 1(I) cDNA. The mismatch was not present in RNA/RNA hybrids from either parent. This region of both alpha 1(I) alleles of the patient was isolated by screening a lambda ZAP cDNA library. Sequence determination of both alleles demonstrated a single nucleotide change, G----A, resulting in the substitution of a serine for a glycine at amino acid residue 832. This point mutation occurs in the coding region for alpha 1(I) CB6 and is concordant with the protein data. The finding of a glycine substitution in an alpha 1(I) chain of a patient with the milder type IV osteogenesis imperfecta phenotype requires modification of current molecular models for types II and IV osteogenesis imperfecta.
