Programmed cell death in normal fetal rat lung development.

Lung development is a coordinated process regulated by the interactions of extracellular and intracellular factors, yet little is known about the process of programmed cell death during lung development. To study this question, we examined fetal rat lung from the pseudoglandular period (gestational day 15) to the day of birth (gestational day 21) using BrdU incorporation into DNA as a proliferative marker, while in parallel examining several markers of programmed cell death including terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), DNA "laddering, " and expression of programmed cell death pathway proteins. Cell proliferation was ongoing throughout fetal days 15 to 21 with a decrease in proliferation over days 20 and 21. Programmed cell death in fetal lung also appeared to be present at all ages examined, but demonstrated 2 peaks of activity at fetal days 15 and 18 to 20. Bcl-XL expression was detected on fetal days 15 to 21, with diminished expression on days E15 to E18. Cleaved poly(ADP-ribose)polymerase (PARP), activated caspase-3, Bax, and Bad were increased on days 18 to 20. We conclude that proliferation is the primary process driving fetal lung development with programmed cell death occurring throughout the lung developmental process to refine structural remodeling.

Regulation of the rat BB1 RNA during normal rat lung development.

BB1 was recently cloned from the WI-38 human fetal lung cell line. Human BB1 (hBB1) is expressed by multiple tissues, including lung. Because inhibition of BB1 translation using antisense oligodeoxynucleotides resulted in prevention of G1 traversal in cultured cells, we hypothesized that BB1 gene expression would be regulated during lung development with greater expression during periods of active lung growth. To gain insight into the expression of BB1 during lung development, a rat BB1 (rBB1) homologue was cloned and used in Northern hybridization analyses and in situ hybridization histochemistry (ISHH). Northern hybridization analyses of fetal and postnatal rat lung demonstrate that rBB1 RNA abundance is relatively low on fetal days E17 through E19, with a small peak of expression occurring on fetal day E20, then increases at birth with peak expression in adult lung. ISHH correlates with the Northern hybridization data and reveals rBB1 RNA expression throughout lung from E17 to E21 in both epithelium and mesenchyme. In postnatal lung, more intense expression of BB1 was observed than in fetal lung, localizing BB1 transcripts to proximal and distal airways and mesenchymal cells surrounding airways. Proliferating cell nuclear antigen (PCNA) was identified in lung sections adjacent to those used for ISHH and it was found that BB1 expression was present in PCNA-positive cells; however, BB1 expression was not limited to PCNA-positive cells in either the fetal or postnatal periods. This was most apparent in adult (60-day) rat lung where essentially no PCNA-positive cells were detected, but intense BB1 expression was detected in airway epithelium and surrounding mesenchyme. These studies demonstrate developmental regulation of BB1 during lung development. The findings are consistent with BB1 action in cell growth-related processes of fetal and early postnatal lung; however, the distribution of BB1 expression in relation to PCNA localization suggests that BB1 participates in cellular functions in addition to cell proliferation.

Regulation of the insulin-like growth factor system during normal rat lung development.

Insulin-like growth factor (IGF)-I and IGF-II are small peptide growth factors that interact with a specific membrane receptor, the type 1 IGF receptor, to stimulate cellular proliferation and/or differentiation. The actions of these growth factors and their availability to their receptors are modulated by specific binding proteins, IGF binding protein (IGFBP)-1 through -6, which together with the IGFs and IGF receptors form the IGF system. We have analyzed RNA extracted from fetal (gestation day 16 [E16] through 22 [E22]) and adult (60-day-old) rat lung for expression of each component of the IGF system. IGF-I and -II RNAs are expressed throughout fetal development. IGF-I mRNA remained relatively constant in fetal and adult lung, whereas IGF-II RNA decreased in later gestation to levels below detection by Northern analyses in adult lung. Type 1 IGF receptor expression varied little through all ages studied, whereas the type 2 IGF receptor RNA displayed developmental regulation with a decline in expression with advancing age. IGFBP-1 transcripts were not detected in fetal or adult lung. IGFBP-2 RNA was expressed from E16 to E22, although its abundance decreased in late gestation and in adult lung, with the lowest levels of expression on day E22. IGFBP-3, -4, and -5 had similar profiles of RNA abundance, with fetuses at ages E21 and E22 displaying higher levels of transcript abundance as compared with those aged E17 to E20; the lowest RNA abundance was seen at E20.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning and characterization of a novel RNA involved in cellular growth regulation.

During the course of antisense oligodeoxynucleotide (oligo) inhibition experiments investigating the role of insulin-like growth factor I (IGF-I) in the WI-38 cell cycle, we found that a sense-strand oligo (S oligo), used as a control, inhibited DNA synthesis 90 to 95%. S1 nuclease protection assays demonstrated that this S oligo formed intracellular duplexes with WI-38 RNA, and Northern (RNA) hybridization analyses demonstrated specific hybridization of this 32P-labeled S oligo to 1.8-, 2.3-, and 3.2-kb RNAs. We have cloned and sequenced a 2,251-bp cDNA, designated BB1, corresponding to the 2.3-kb RNA. Decoding of the BB1 cDNA sequence reveals several open reading frames arranged in a motif similar to that seen in proteins subject to translational control mechanisms. Homology searches of nucleic acid and protein data bases reveal no significant homology of BB1 with known sequences other than a 234-bp region in the BB1 5' untranslated region that shared 97% homology with a region in the 3' untranslated region of the human cdc42 mRNA. S1 nuclease protection analyses performed with IGF-I gene fragments and computer homology searches demonstrated that the BB1 RNA does not derive from transcription from the opposite strand of the IGF-I gene. Northern hybridization analyses of RNA extracted from serum-starved HeLa S3 cells demonstrated that steady-state BB1 RNA levels increased upon serum growth stimulation, with steady-state levels peaking 4 h after release from the block induced by serum starvation. Antisense oligo inhibition experiments using specific BB1 antisense oligos targeted to the putative open reading frames of the BB1 RNA reduce DNA synthesis of HeLa S3 cells to 15% of control levels, indicating that the BB1 RNA is essential for cell cycle traversal and, as such, encodes a growth-reguLating gene product.

Insulin-like growth factor-I (IGF-I) antisense oligodeoxynucleotide mediated inhibition of DNA synthesis by WI-38 cells: evidence for autocrine actions of IGF-I.

Insulin-like growth factor-I (IGF-I) is elaborated into culture medium by WI-38 cells, a human embryonic lung fibroblast cell line, and may participate in the autocrine stimulation of DNA synthesis. We have confirmed the expression of IGF-I by these cells and documented that they express the type 1 IGF receptor and a number of IGF-binding proteins. In situ hybridization histochemistry demonstrated relatively uniform expression of IGF-I and type 1 IGF receptor transcripts among WI-38 cells. To determine whether WI-38-synthesized IGF-I exerted mitogenic effects, a 15-base oligodeoxynucleotide complementary to the 5'IGF-I mRNA sequence (IGF-I AS-Oligo), including the translation start site, was incubated with cultured cells in an attempt to inhibit IGF-I synthesis. The IGF-I AS-Oligo was stable in cell culture, formed intracellular duplexes with IGF-I mRNA, and at 2 microM reduced IGF-I in conditioned medium by 83%. The IGF-I AS-Oligo also inhibited [3H]thymidine incorporation into DNA in a dose-dependent fashion (by 77% at 2 microM and by 95% at 20 microM). This reduction in DNA synthesis was prevented when the medium was supplemented with 100 ng/ml IGF-I. The oligomer also decreased the abundance of IGF-binding proteins in conditioned medium. The IGF-I AS-Oligo appears to exert its effects by blocking IGF-I mRNA translation, rather than blocking transcription or initiating RNase-H activity, because the abundance of IGF-I transcripts was not decreased in its presence. These findings confirm an essential role for IGF-I in WI-38 cell DNA synthesis and are consistent with autocrine actions by WI-38 cell IGF-I.

Characterization of antibodies to idiotypic determinants of monoclonal anti-sperm antibodies.

Rabbit polyclonal antibodies to the idiotype of murine monoclonal anti-sperm antibodies were developed and characterized. M29.6 and M42.15 are monoclonal antibodies (mAbs) that inhibit fertilization in vivo and in vitro. Sera from rabbits inoculated with purified mAbs (Ab1) were absorbed with normal mouse and isotype-specific immunoglobulin (Ig); the anti-idiotype Ig fraction (Ab2) was isolated by protein A-chromatography or by chromatography on the corresponding idiotype column. Binding specificity of Ab2 was confirmed by measuring the reactivity of Ab2 with homologous and heterologous mAbs. Ab2 competitively inhibited 125I-labeled Ab1 binding to mouse sperm, suggesting that the Ab2 preparation possessed subpopulations directed against idiotopes similar or adjacent to the antigen-binding site of the mAb. Anti-idiotype antibodies reactive with the antigen-combining site of the anti-sperm mAb may contain subpopulations that mimic the mouse sperm epitope recognized by Ab1. Immunization with Ab2 induced anti-(anti-idiotype) antibodies (Ab3), which competitively inhibited binding of 125I-labeled Ab1 to immobilized Ab2. These results are consistent with the hypothesis that immunization of mice with antibodies to the idiotype of sperm-specific mAbs can induce antibodies that share structural similarities with the anti-sperm mAb used for their induction. Immunization with anti-idiotype antibodies that mimic sperm antigen structure represents a possible strategy for induction of immunity to sperm.