A role for chloride in the hyperpolarizing effect of acetylcholine in isolated frog vestibular hair cells.

Acetylcholine (ACh) is the dominant transmitter released from inner ear efferent neurons. In frog vestibular organs, these efferent neurons synapse exclusively with type II hair cells. Hair cells isolated from the frog saccule hyperpolarize following the application of 50 microM ACh, thereby demonstrating the presence of an ACh receptor. A role for Cl(-) in the response of hair cell-bearing organs to efferent nerve activation or ACh application was suggested some years ago. Perfusion with solutions in which most of the Cl(-) was replaced by large impermeant anions decreased the cholinergic inhibition of afferent firing in the cat and turtle cochleas, and frog semicircular canal. Our previous work in the intact organ demonstrated that substitution of large impermeant anions for Cl(-) or use of Cl(-) channel blockers reduced the effect of ACh on saccular afferent firing. Using the perforated-patch clamping technique, replacement of Cl(-) by methanesulfonate, iodide, nitrate, or thiocyanate attenuated the hyperpolarizing response to ACh in hair cells isolated from the frog saccule. The chloride channel blockers picrotoxin and 4,4'-dinitrostilbene-2,2'-disulfonic acid were also tested and found to inhibit the ACh response. Thus, the present work demonstrates that the effects of Cl(-) substitutions or Cl(-) channel blockers on the ACh response in the intact saccule can be explained completely by effects on the hair cell. Evidence is also presented for the presence of the messenger RNA for a calcium-dependent chloride channel in all hair cells but especially saccular hair cells. This channel may be involved in the response to ACh. The precise role for chloride in this response, whether as a distinct ion current, as a transported ion, or as a permissive ion for other components, is discussed.

Lung-specific expression in mice of a dominant negative mutant form of the p53 tumor suppressor protein.

Lung cancer is the most frequent cause of cancer deaths in the United States. A strong correlation exists between mutations in the gene encoding the p53 tumor suppressor protein and lung malignancies. Our goal is to prepare a transgenic mouse model with disrupted p53 function in the epithelial cells of the peripheral lung. To achieve this goal, a "dominant negative" mutant form of p53 was expressed from the human surfactant protein C (SPC) promoter. The dominant negative p53 expressed from the SPC promoter will antagonize wild-type p53 functions in alveolar type II pneumocytes and some bronchiolar cells of the transgenic animals and thereby promote development of carcinoma of the lung. This animal model should prove useful to the study of lung carcinogenesis and to the identification of agents that contribute to neoplastic conversion in the lung.

Genetic determinants of feline leukemia virus-induced multicentric lymphomas.

Three discrete forms of feline leukemia virus (FeLV)-associated lymphoma have been described clinically: (1) thymic, (2) alimentary, and (3) multicentric. The most common and best-characterized lymphomas are of T-cell origin, generally occurring in the thymus. These tumors typically contain mature T-cells, involve the activation of a distinctive set of proto-oncogenes, and contain FeLV proviruses whose long terminal repeat (LTR) sequences contain tandemly repeated enhancers. Previous studies of a small group of extrathymic, multicentric lymphomas implicated a different set of genetic determinants. The present study expands those observations by examining the lineage of origin, the involvement of proto-oncogenes, and the structure of LTR and env gene sequences in a set of 11 natural, extrathymic lymphomas of the multicentric type. A pattern of genetic events associated with FeLV-positive multicentric lymphomas emerges from this analysis that is clearly distinct from the pattern associated with thymic lymphomas. The tumors do not contain T-cells or B-cells, as evidenced by the germ line organization of TCR beta and IgH loci. Proto-oncogenes strongly implicated in T-cell lymphomagenesis are not involved in these tumors. Rather, a distinct set of proto-oncogenes may be involved. Most striking is the repeated occurrence of an FeLV isolate whose LTR and env gene bear unique sequence elements.

Function of a unique sequence motif in the long terminal repeat of feline leukemia virus isolated from an unusual set of naturally occurring tumors.

Feline leukemia virus (FeLV) proviruses have been characterized from naturally occurring non-B-cell, non-T-cell tumors occurring in the spleens of infected cats. These proviruses exhibit a unique sequence motif in the long terminal repeat (LTR), namely, a 21-bp tandem triplication beginning 25 bp downstream of the enhancer. The repeated finding of the triplication-containing LTR in non-B-cell, non-T-cell lymphomas of the spleen suggests that the unique LTR is an essential participant in the development of tumors of this particular phenotype. The nucleotide sequence of the triplication-containing LTR most closely resembles that of FeLV subgroup C. Studies performed to measure the ability of the triplication-containing LTR to modulate gene expression indicate that the 21-bp triplication provides transcriptional enhancer function to the LTR that contains it and that it substitutes at least in part for the duplication of the enhancer. The 21-bp triplication confers a bona fide enhancer function upon LTR-directed reporter gene expression; however, the possibility of a spacer function was not eliminated. The studies demonstrate further that the triplication-containing LTR acts preferentially in a cell-type-specific manner, i.e., it is 12-fold more active in K-562 cells than is an LTR lacking the triplication. A recombinant, infectious FeLV bearing the 21-bp triplication in U3 was constructed. Cells infected with the recombinant were shown to accumulate higher levels of viral RNA transcripts and virus particles in culture supernatants than did cells infected with the parental type. The triplication-containing LTR is implicated in the induction of tumors of a particular phenotype, perhaps through transcriptional regulation of the virus and/or adjacent cellular genes, in the appropriate target cell.

Retroviral determinants of leukemogenesis.

The slowly transforming, leukemogenic retroviruses of humans and other mammals induce malignant disease after prolonged latency but lack an oncogene to which their malignant potential can be attributed directly. The leukemogenic activity of these retroviruses can be attributed to at least three factors, including (1) transcriptional regulatory sequences in the long terminal repeat: (2) the insertional mutagenesis of cellular protooncogenes, thus activating their malignant potential; and (3) the actions of structural and regulatory proteins encoded by viral genes. The goal of this review is to summarize recent findings regarding the roles of these factors in retroviral leukemogenesis. The focus of the review is on the slowly transforming, leukemogenic retroviruses of mammals, including humans and experimental animals.