Implication of alpha4 phosphoprotein and the rapamycin-sensitive mammalian target-of-rapamycin pathway in prolactin receptor signalling.

A prolactin (PRL)-responsive 3'-end cDNA encoding rat alpha4 phosphoprotein was previously isolated from a rat lymphoma cDNA library. Rat alpha4 is a homologue of yeast Tap42 and is a component of the mammalian target-of-rapamycin (mTOR) signalling pathway that stimulates translation initiation and G1 progression in response to nutrients and growth factors. In the present study, the full-length rat alpha4 cDNA was obtained by 5'-RACE and the 1023 bp open reading frame predicted a 340 amino acid protein of 39.1 kDa. The alpha4 mRNA was expressed in quiescent PRL-dependent Nb2 lymphoma cells deprived of PRL for up to 72 h but expression was downregulated within 4 h of PRL treatment. In contrast, PRL-independent Nb2-Sp cells showed constitutive expression of alpha4 that was not affected by PRL. Western analysis of Nb2 cell lysates or of V5-tagged-alpha4 expressed in COS-1 cells detected a single immunoreactive band of approximately 45 kDa. Enzymatic deglycosylation of affinity-purified 45 kDa alpha4 yielded the predicted 39 kDa protein. Phosphorylation of Nb2 alpha4 was induced by PRL or 2-O-tetradecanoyl-phorbol-13-acetate (TPA) and further enhanced by a combination of PRL and TPA. The Nb2 alpha4 associated with the catalytic subunit of protein phosphatase 2A and localized predominantly in Nb2 nuclear fractions with trace amounts in the cytosol. The immunosuppressant drug rapamycin inhibited proliferation of Nb2 cells in response to PRL or interleukin-2, but had no effect on Nb2-Sp cells. Furthermore, transient overexpression of alpha4 in COS-1 cells inhibited PRL stimulation of the immediate-early gene interferon regulatory factor-1 promoter activity. Therefore, PRL downregulation of alpha4 expression and/or PRL-inducible phosphorylation of alpha4 may be necessary for PRL receptor (PRLr) signalling to the interferon regulatory factor-1 promoter in the Nb2 cells and, furthermore, implicates cross-talk between the mTOR and PRLr signalling cascades during Nb2 cell mitogenesis.

Prolactin, interleukin-2 and FGF-2 stimulate expression, nuclear distribution and DNA-binding of rat homolog of pombe Cdc5 in Nb2 T lymphoma cells.

Pombe and human Cdc5 have been implicated in G2/M progression, but recently Cdc5 was identified as a component of a multiprotein complex essential for pre-mRNA splicing. We have previously isolated a prolactin (PRL)-inducible partial cDNA (1907 bp) encoding rat Cdc5. In the present study, the full length rCdc5 sequence (2847 bp) was obtained by 5'-RACE and cytokine regulation of Cdc5 expression was examined. PRL and interleukin-2 (IL2) act as mitogens in Nb2 T-lymphoma cells. Fibroblast growth factor (FGF-2) is not mitogenic in Nb2 cells but inhibits apoptosis of PRL-deprived cells. This study showed that PRL, IL-2 and FGF-2 rapidly increased Nb2 Cdc5 expression (3.4 kb mRNA) to reach 2-3-fold above controls at 4 h, and Cdc5 mRNA levels remained elevated at 24 h. There was a corresponding 2-3-fold increase in Cdc5 protein (105 kDa) levels at 24 h. Immunoblotting and fluorescent confocal microscopy showed predominant nuclear/perinuclear Cdc5 in quiescent Nb2 cells. PRL or FGF-2 treatment transiently increased nuclear Cdc5-specific immunofluorescence at 4 h but IL-2 gave maximal nuclear accumulation of Cdc5 at 24 h. The deduced rCdc5 protein has approximately 98% amino acid identity with human Cdc5. Like other Cdc5 family members, the N-terminus of rCdc5 contains two repeats of a DNA-binding domain found in a-, b- and c-Myb. Gel shift assays using (32)P-labeled Myb consensus oligonucleotides revealed two Myb-specific DNA-protein complexes in Nb2 nuclear extracts. Formation of both complexes was increased by PRL or FGF-2 at 1-5 and at 20 h and was partially inhibited by anti-Myb or anti-Cdc5 antibodies. In summary, rapid activation of Cdc5 in response to mitogenic and non-mitogenic stimuli suggests a complex role for Cdc5 in cellular regulation and this may not be restricted to mitotic entry or G2/M progression as previously supposed.

Identification and nuclear localization of a novel prolactin and cytokine-responsive carboxypeptidase D.

A full-length, PRL-inducible complementary DNA (cDNA) encoding a novel, nuclear-targeted carboxypeptidase D isoform (designated CPD-N) was identified in the rat PRL-dependent Nb2-11C and PRL-independent Nb2-Sp lymphoma cell lines by differential display. The CPD-N cDNA (3751 bp) has 99% (3582/3583) homology with rat carboxypeptidase D (CPD; 4377 bp). In comparison to the rat CPD cDNA (ORF of 4134 bp; 180-kDa protein), CPD-N was shorter by approximately 600 bases but contained 148 unique bases at the 5'-end to give an ORF of 3399 bp. RT-PCR with primers specific to the 5'-end of CPD-N or to CPD showed that the CPD-N transcript was expressed in the Nb2-11C and Nb2-Sp cells but was not detected in rat brain or lung. Conversely, the CPD transcript was expressed in rat brain but was not detected in the two Nb2 cell lines. CPD-N expression (7.5-kb messenger RNA) was stimulated by PRL (10 ng/ml) and/or by interleukin-2 (24 U/ml) in Nb2-11C and Nb2-Sp cells. Most rat tissues expressed multiple CPD transcripts (7.5, 4.1, and 2 kb). Curiously, CPD transcripts were low or undetectable in male rat liver but readily detected in female liver, suggesting that sex-specific hormone levels may regulate its expression. Indeed, CPD expression in the PRL-responsive HepG2 hepatoma and MCF-7 breast cancer cell lines was low in control cells but was markedly stimulated by PRL after 3 h. Consistent with the shorter ORF of CPD-N, Western analysis detected proteins of smaller molecular sizes of 160 kDa (abundant) and 117 kDa (weak) in the Nb2-11C cells. The Nb2-Sp cells expressed a single and abundant 117-kDa protein, implicating differential protein processing in the two cell lines. Rat CPD has been reported to colocalize with the trans-Golgi network marker TGN38. Subcellular fractionation showed predominant nuclear localization of CPD-N and trace amounts were detected in the 100,000 x g microsomal fraction after PRL treatment (4 h); in contrast, TGN38 was found only in the microsomal fraction at this time. In cells treated with PRL for 24 h, immunofluorescent confocal microscopy showed nuclear and cytoplasmic distribution of CPD-N. Cytoplasmic CPD-N colocalized with TGN-38 whereas nuclear CPD-N had a mesh-like distribution and colocalized with nuclear lamin B.

Fibroblast growth factor-2 stimulates endothelial nitric oxide synthase expression and inhibits apoptosis by a nitric oxide-dependent pathway in Nb2 lymphoma cells.

We recently reported that the rat Nb2 T lymphoma cells expressed messenger RNAs (mRNAs) encoding both fibroblast growth factor-2 (FGF-2) and the FGF receptor, suggesting possible paracrine and/or autocrine roles for FGF-2 in lymphoma cell function. We have also shown that the Nb2 cells expressed endothelial nitric oxide synthase (eNOS) and produced low levels of nitric oxide (NO) that inhibited apoptosis of PRL-deprived cells via a PRL-independent, bcl-2-mediated pathway. In this study the effects of PRL and FGF-2 on Nb2 cell survival and NO production were further investigated. The percentages of nonapoptotic cells in PRL-treated vs. PRL-deprived cultures after 6 days were 95% and 53%, respectively. Addition of FGF-2 to PRL-deprived Nb2 cells did not stimulate cell proliferation, but the onset of apoptosis was significantly inhibited, such that more than 85% of the cells remained nonapoptotic after 6 days. The steady state levels of bcl-2 and bag-1 mRNAs were low in PRL-deprived Nb2 cells, but were markedly increased by PRL or FGF-2. bcl-2 expression was induced within 1 h of PRL or FGF-2 addition and continued to increase to a level 20- to 25-fold above the control level within 24 h. bag-1 expression also increased within 1 h after the addition of PRL or FGF-2, was maximal within 8 h, and declined slowly thereafter. The levels of eNOS mRNAs were low but detectable in growth-arrested Nb2 cells, and PRL further down-regulated eNOS mRNA levels over the next 24 h. In contrast, FGF-2 significantly increased eNOS mRNA levels within 2 h to reach a peak 10-fold induction by 12 h. FGF-2 stimulation of eNOS mRNA was accompanied by a 2- to 3.5-fold increase in cellular levels of the eNOS protein and a 2.5-fold increase in serine-phosphorylated eNOS. However, the ratio of serine-phosphorylated eNOS vs. total cellular eNOS was unchanged, indicating that FGF-2 did not affect the serine phosphorylation status of eNOS. Nb2 cells produced low basal levels of NO, which increased with increasing L-arginine concentrations. PRL did not further increase NO release in the presence of L-arginine (0.1 or 1 mM), but FGF-2 significantly (P:

L-arginine inhibits apoptosis via a NO-dependent mechanism in Nb2 lymphoma cells.

Prolactin (PRL) inhibits apoptosis and stimulates proliferation of the PRL-dependent rat Nb2 lymphoma cell line by divergent signaling pathways. Nitric oxide (NO) was recently identified as a downstream regulator of PRL action, and as an inhibitor of apoptosis in immune cells. In the present study, the role of NO in PRL-regulated Nb2 cell function was investigated. Nb2 cells expressed the endothelial nitric oxide synthase (eNOS) isoform, whereas neuronal NOS (nNOS) and inducible NOS (iNOS) mRNAs were undetectable. The eNOS mRNA was abundantly expressed in PRL-deprived, growth-arrested cells but decreased by at least 3-fold at 3-24 h following PRL treatment. Downregulation of eNOS was not accompanied by a corresponding decrease in the eNOS protein, the level of which remained constant for at least 24 h after PRL treatment. PRL had no effect on the phosphorylation state or subcellular redistribution of the eNOS enzyme, or on production of NO by Nb2 cells. However, increasing concentrations of L-arginine (NOS substrate) alone increased NO production in these cells and significantly enhanced PRL-stimulated cell proliferation. NO releasers (SNAP, DEA/NO, SIN-1) also significantly enhanced Nb2 cell proliferation in the presence of a submaximal dose of PRL (0.125 ng/ml). In the absence of PRL, the NO releasers alone promoted cell survival and maintained a viable cell density significantly higher than that of untreated PRL-deprived cells. L-arginine or the NO releaser DEA/NO alone significantly inhibited apoptosis in Nb2 cells deprived of PRL for 5 days. Expression of the anti-apoptotic gene bcl-2, which was stimulated within 1 h by PRL, was upregulated by L-arginine or DEA/NO alone at 2 h and 8 h, respectively. These findings suggest that NO produced by eNOS inhibits apoptosis and promotes the survival of growth-arrested Nb2 lymphoma cells via a prolactin-independent, Bcl-2-mediated pathway.

Estrogen stimulates expression of adenine nucleotide translocator ANT1 messenger RNA in female rat hearts.

The identification of estrogen-responsive genes in the heart, is necessary to understand estrogen-induced changes in cardiac function. Using Delta RNA fingerprinting, we demonstrate that a single injection of estradiol benzoate (50 microg, s.c.) revealed mRNA species that were elevated, down-regulated, or were unaffected in the heart tissue of ovariectomized female rats. One of the upregulated genes was identified, by cloning and sequencing, to have 95.8% (230/240) identity with the 3' end of the rat ant1 gene encoding the mitochondrial adenine nucleotide translocator, ANT1. Using the isolated ANT1 cDNA (280 bp) as a probe in Northern analysis, estrogen was shown to upregulate the expression of cardiac ANT1, by at least 3-fold in female rats, from as early as 1 h to as long as 24 h. In contrast, estrogen treatment had no effect on ANT1 expression in heart tissue from male rats. RNA yields were low in rat atria and no transcript was detectable by Northern analysis. Using primers specific to the known rat ANT1 gene, the estrogen upregulation of the cardiac ANT1 transcript in female rat was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR); a predicted product of 249 bp was obtained and this was stimulated by at least 3-fold upon estrogen treatment for 24 h.

Malignant progression of rat Nb2 lymphoma cells: chromosomal alterations and metastatic properties.

Cultured rat pre-T Nb2 lymphoma cell lines have provided a useful model for tumor progression of T-cell cancers. Comparative analysis of the non-metastatic, prolactin (PRL)-dependent parental Nb2-U17 line and its PRL-independent and/or metastatic sublines, can be used in a search for progression-related genomic alterations. In the present study, the PRL-dependent, cloned Nb2-11C and PRL-independent Nb2-Sp sublines were used to examine development of metastatic ability and PRL independence relative to chromosomal alterations. Metastatic ability was determined using Noble rats carrying subcutaneous tumor transplants; PRL dependence/autonomy was checked in culture. Nb2-11C tumor transplants quickly gave rise to morbidity, associated with metastases in kidney and liver. Transplants of the slower growing Nb2-Sp cells showed variable tumorigenicity as metastases developed in only 40% of the rats (in lungs, kidney, stomach). G-banded chromosome analysis showed the Nb2-11C culture had the karyotype of the parental Nb2-U17 line plus an extra chromosome 19, thus, indicating an association between the development of metastatic ability in Nb2-11C cells and trisomy 19. The Nb2-Sp subline was not clonal. Its stemline showed two alterations in the parental karyotype: acquisition of an add(7)(q10) and loss of the extra chromosome add(15)(p12). Additional abnormalities, add(6)(q11) and trisomy 19, occurred in 15% and 5% of the Nb2-Sp population, respectively. Passaging of the Nb2-Sp subline in vivo resulted in generation and/or outgrowth of new sublines, a major one of which showed an apparent transient growth requirement for lactogens. Possible mechanisms underlying the PRL independence and in vivo properties of the Nb2-Sp cells are discussed.

Prolactin induces expression of FGF-2 and a novel FGF-responsive NonO/p54nrb-related mRNA in rat lymphoma cells.

The rat Nb2-11C lymphoma cell line expresses high affinity prolactin (PRL) receptors, and requires lactogenic hormones for survival and proliferation. We have applied differential display to identify genes which are differentially induced in Nb2-11C cells following PRL stimulation, or which are constitutively expressed in the PRL-independent Nb2-Sp cells. In the present study we characterized a clone (22c.2) which was expressed in Nb2-Sp cells, and in Nb2-11C cells given PRL for 3 h but not in untreated cells. The 279 bp cDNA had 95% homology with the 3' end of the murine 2.6 kb FGF-inducible gene 14 (FIN14). When clone 22c.2 was used to screen a Nb2-Sp cDNA library to obtain a longer cDNA, a unique 1039 bp clone PNR (Prolactin-responsive/ NonO-Related) was isolated, subcloned and sequenced. The deduced amino acid sequence encoded by the PNR open reading frame had significant homology with a family of RNA- and DNA-binding proteins which include the human polypyrimidine tract-binding protein (PTB)-associated splicing factor (PSF), the murine non-POU-domain-containing octamer-binding protein (NonO) and the human NonO homologue p54nrb. Nb2-11C cells expressed three PNR-related mRNA transcripts of 2.5, 3.0 and > 10 kb. Expression of the 2.5 and 3.0 kb transcripts were increased at least 4-fold within 3 h of PRL treatment. PNR expression was also significantly stimulated within 3 h by addition of FGF-2 to either Nb2-11C or Nb2-Sp cells, although alone FGF-2 was not mitogenic for either cell line. Reverse transcription-polymerase chain reaction (RT-PCR) confirmed the expression of both FGF-2 and FGF receptor mRNA in Nb2 cells. raising the possibility of an autocrine or paracrine function for FGF-2 in lymphoma cells. Furthermore, PRL rapidly stimulated the expression of FGF-2 mRNA in a time- and dose-dependent manner in both Nb2-11C and Nb2-Sp cells. FGF-2 expression was increased within 1 h and was maintained at a high level for at least 10 h following treatment with 2 ng/ml PRL. Western blotting with anti-FGF2 antisera demonstrated PRL stimulation of intracellular accumulation, but not secretion of immunoreactive FGF-2. The observation of PRL-responsive expression of FGF-2 in Nb2 cells suggests a previously unrecognized pathway for PRL action in lymphoid cells.

Rapid induction of Jak2 and Sp1 in T cells by phosphorothioate oligonucleotides.

Phosphorothioate-modified ODNs ([S]ODNs) are known to exert a variety of sequence-independent effects that are mediated in part by rapid induction of the Sp1 transcription factor. An unidentified tyrosine kinase was implicated in this Sp1 induction. In the present study, antisense [S]ODNs, initially designed to target three signaling molecules in the prolactin (PRL)-responsive rat Nb2 T cell line rapidly elevated Jak2 tyrosine kinase and Sp1 protein levels. The [S]ODN-mediated elevation of Jak2 peaked (3-fold to 6.5-fold above controls) at 15 minutes and returned to basal levels by 1 hour, whereas elevation of Sp1 (about 2-fold above controls) peaked at 1 hour. The [S]ODN-mediated induction of Sp1, but not Jak2, was abrogated by AG 490, a Jak2-specific inhibitor. In the presence of submaximal doses of PRL (0.18-0.36 ng/ml), [S]ODN-mediated induction of Jak2 and Sp1 was sustained for 72 hours. Furthermore, the [S]ODNs alone significantly increased Nb2 cell growth and enhanced the growth stimulatory effects of PRL on these cells. In contrast, unmodified ODNs had no effect on Jak2 or Sp1 protein levels and did not stimulate Nb2 cell growth. In conclusion, [S]ODNs stimulate the coordinate induction of Jak2 and Sp1 and stimulate Nb2 T cell proliferation in a sequence-independent manner. The abrogation of Sp1 induction by AG 490 indicates that Jak2 tyrosine kinase is required for [S]ODN-mediated induction of Sp1 in these cells. These results may help to explain some of the nonspecific effects of [S]ODNs, particularly in cytokine-dependent immune cells.

A novel mutation in Exon 4 of the low density lipoprotein receptor gene resulting in heterozygous familial hypercholesterolemia associated with decreased ligand binding.

Familial hypercholesterolemia (FH) is an autosomal dominant disorder caused by mutations in the low density lipoprotein (LDL) receptor gene. Currently, diagnosis of heterozygous FH relies on clinical phenotype; however, the use of clinical criteria for the diagnosis of heterozygous FH does not always permit unequivocable diagnosis of the disease. Molecular diagnosis of FH is clinically valuable especially in regions where founder mutations exist or where polygenic hypercholesterolemia is prevalent. In this paper we report the identification of a novel mutation, a cytosine to guanine substitution, at codon 152 in exon 4 of the LDL receptor gene in a Nova Scotian family clinically diagnosed with heterozygous FH. The mutation creates a recognition sequence for the restriction endonuclease BsrI, and can be readily detected by BsrI restriction analysis of a 160 bp amplicon spanning the mutation. This analysis was used to show that the mutation segregated with the disease in this family and is the probable cause of FH in this kindred.

FGF-2 antisense RNA encodes a nuclear protein with MutT-like antimutator activity.

Bidirectional transcription of the basic fibroblast growth factor (FGF-2) gene gives rise to multiple polyadenylated sense mRNAs and a unique 1.5 kb antisense transcript (FGF-AS) which is complementary to the 3'-untranslated region of the FGF-2 mRNA. The rat FGF-AS cDNA encodes a novel 35 kDa nuclear protein (GFG) with homology to the MutT family of antimutator NTPases. Antibodies against the deduced amino acid sequence of GFG detected intense immunoreactivity in the nuclei of adult rat hepatocytes. Subcellular fractionation and Western blotting confirmed the presence of a 35 kDa immunoreactive protein in the nuclear fraction and, to a lesser extent, in the mitochondrial fractions of rat liver homogenates. Recombinant GFG suppressed the spontaneous mutation rate of MutT-deficient E. coli in a complementation assay. In-frame deletion of the 53 amino acids encompassing the MutT domain eliminated this activity, confirming the catalytic function of this region in the FGF antisense gene product. These findings demonstrate for the first time that the FGF-AS transcript encodes a functional nuclear protein with MutT-related enzymatic activity.

Differential expression of elongation factor-2, alpha4 phosphoprotein and Cdc5-like protein in prolactin-dependent/independent rat lymphoid cells.

The differential display of mRNAs technique was used to identify genes which are differentially expressed in the prolactin (PRL)-dependent Nb2-11C and PRL-independent Nb2-Sp rat lymphoma cell lines. The technique was validated by the differential display of the proto-oncogene c-myc in PRL-treated, but not in untreated, Nb2-11C cells. Nine DNA bands, isolated from the 6% display gels and confirmed by reverse Northerns to be differentially expressed, were cloned and gave rise to ten unique clones. DNA sequencing showed that these clones had limited homologies to known genes or uncharacterized expressed sequence tags. Using one of these clones (6ac1.12) as a probe in Northern analysis, a transcript of approximately 4 kb was detected which was elevated in PRL-treated Nb2-11C cells and in mid-log phase growing Nb2-Sp cells. Similar to c-myc, expression of the 4 kb transcript increased in Nb2-11C cells given PRL for 3 h (+/- the phorbol ester TPA) but not in cells given TPA alone. The 4 kb transcript also increased with increasing Nb2-11C cell densities. By screening an Nb2-Sp cDNA library with 6ac1.12 as probe, three unique genes were isolated and identified as elongation factor 2 (EF-2), alpha4 phosphoprotein and a Cdc5-like protein. Each of the three genes were PRL responsive in Nb2-11C cells and expressed constitutively in Nb2-Sp cells. The expression of EF-2 or alpha4, but not the Cdc-like protein, was dependent on cell densities. EF-2 regulates protein synthesis while the alpha4 and Cdc5-like phosphoproteins have been implicated in IgG receptor-mediated and mitogen-activated signaling, respectively. The identification that these genes are PRL-responsive and/or differentially expressed in the Nb2-11C and Nb2-Sp cell lines may permit insights into the molecular changes that are involved in regulating the transition to growth factor independence in lymphoid tumors.

Induction of Sp1 activity by prolactin and interleukin-2 in Nb2 T-cells: differential association of Sp1-DNA complexes with Stats.

The induction of the transcription factor Sp1 by prolactin (PRL) and interleukin-2 (IL-2) was investigated in the PRL- and IL-2 responsive rat Nb2 T-cell line. Western analysis showed a rapid increase in Sp1 synthesis in Nb2 cells in response to PRL or IL-2. Elevation of Sp1 protein levels occurred within 15 min following PRL or IL-2 stimulation, reached a maximum by 1 h and was inhibited by cycloheximide, indicating de novo protein synthesis. Interestingly, dilution of confluent, growth-arrested Nb2 cells to low density also caused a rapid elevation in Sp1 suggesting that growth arrest may down-regulate Sp1 synthesis. Electrophoretic mobility shift assays using an Sp1 consensus oligonucleotide as probe showed a rapid but transient formation of a single PRL-inducible complex at 30 min. In contrast, three IL-2-inducible complexes were formed at 30 min and persisted to at least 60 min. Mobility shift interference assays using specific Stat antibodies failed to detect Stat1alpha, Stat3 or Stat5 in the 30 min PRL-inducible complex. In contrast, the IL-2 induced complexes contained Stat3 alone at 30 min and both Stat3 and Stat5 at 60 min. The PRL- and IL-2-inducible complexes did not contain the tumor suppressor protein, p53. The time dependent association of the Stat proteins with the IL-2-inducible complexes, but not with the PRL-inducible complex, suggests that the two mitogens may selectively utilize specific promoter elements for transcriptional activation of PRL- and IL-2-responsive genes. Alternatively, the two mitogens may be activating different genes with Sp1-binding promoter elements for their mitogenic action in Nb2 cells.

The basic fibroblast growth factor (FGF-2) antisense RNA (GFG) is translated into a MutT-related protein in vivo.

The basic fibroblast growth factor (FGF-2) gene is transcribed bidirectionally to yield multiple sense (coding) transcripts and a unique 1.5 kb antisense transcript which may regulate sense RNA stability. The antisense RNA also contains a long open reading frame that predicts a hypothetical protein with homology to the prokaryotic MutT antimutator proteins. However, translation of this protein has not previously been demonstrated. We employed antibodies against the conserved MutT-domain of the deduced human FGF-2 antisense protein (GFG) to demonstrate expression of an immunoreactive 24 kDa protein in liver extracts from Xenopus laevis, and two proteins of 28 and 35 kDa in rat liver. In rats, GFG protein expression detected by western blot was tissue-specific and correlated with the level of FGF-2 antisense mRNA expression. These findings demonstrate that, in addition to its possible RNA regulatory function, the FGF-2 antisense transcript is translated into a conserved MutT-related protein.

Passage of X-ray-induced immortal, non-transformed phenotype by DNA-mediated transfection.

To better understand the molecular basis of X-ray-induced carcinogenesis, the immortalization step of this multistep process was examined. Primary rat embryo cells were X-irradiated in vitro and six clones were isolated. Three of these, one designated X-REF-23, were immortal and non-transformed. Transfection of high molecular weight DNA from rat X-REF-23 cells into primary mouse cells yielded two immortal and non-transformed mouse clones, 1K and 2I. Using a 2.3 kb rat-specific repetitive sequence as probe, 1K and 2I were demonstrated to contain rat DNA. This transfected DNA was not any of the known immortalization-associated proto-oncogenes. DNA from 1K was then transfected into primary mouse cells, with or without co-transfection of pSV2 neo DNA. Six immortal mouse clones were isolated and confirmed to contain rat sequences. In conclusion, the immortal phenotype can be transferred by DNA transfection.

Detection of FMRFamide-like immunoreactivities in the sea scallop Placopecten magellanicus by immunohistochemistry and western blot analysis.

FMRFamide-like immunoreactivity was detected histochemically in the sea scallop Placopecten magellanicus. Most immunoreactivity was concentrated in the cerebral, pedal, and parietovisceral ganglia, particularly in the cortical cell bodies and in their fibers which extend into the central neuropile. Whole-mount immunofluorescence studies were used to localize concentrations of immunoreactive cells on the dorsal and ventral surfaces of each ganglion. Immunoreactivity was also detected in nerves emanating from the ganglia. Strong immunoreactivity was localized in peripheral organs, including the gut and gills of juvenile and adult scallops. Weak immunoreactivity was detected in the gonads, heart, and adductor muscle of the adults. A broad FMRFamide-like immunoreactive band of 2.5-8.2 kDa was detected by Western blotting of acetone extracts of the parietovisceral ganglia. In the presence of protease inhibitors, two FMRFamide-like immunoreactive bands (7.2-8.2 kDa and > 17 kDa) were obtained. Neither of these bands comigrated with the FMRFamide standard. It is concluded that peptides of the FMRFamide family are probably regulators of numerous central and peripheral functions in P. magellanicus.

Western blotting of formaldehyde-fixed neuropeptides as small as 400 daltons on gelatin-coated nitrocellulose paper.

A method is described for Western blotting of peptides as small as 400 daltons (Da). Peptides were separated by tricine-sodium dodecyl sulfate electrophoresis and electroblotted to gelatin-coated PH79 nitrocellulose paper (0.1 micron). The electroblotted peptides were fixed to the nitrocellulose paper for 5-10 min in 4% paraformaldehyde solution. Using anti-rabbit FMRF-amide (Phe-Met-Arg-Phe-NH2) as primary antibody, positive immunoreactivity was detected with an amplified alkaline phosphatase assay which was sensitive to at least 0.5 microgram FMRFamide/lane. When immunoreactivity was determined with 125I-protein A, it was possible to amplify and detect weak signals by increasing the autoradiography time. Therefore, using the 125I-protein A detection method, Western blot analysis of brain extracts from Lymnaea stagnalis (pond snail) and Poecilia reticulata (guppy) indicated the presence of four FMRFamide immunoreactive bands after a 7-day exposure to X-ray film. The most abundant peptide coelectrophoresed with the FMRFamide standard (M(r) 598.8 Da). In addition, this Western blotting procedure also detected APGWamide (Ala-Pro-Gly-Try-NH2; 428.5 Da) and [D-Ala2]-Leu-enkephalinamide (568.7 Da) with their respective specific antibodies.

Elevation of the thyroid hormone receptor erb A-alpha 2 mRNA in transformed rodent cells is due to increased message stability.

Previously, the authors reported an elevation of erb A-alpha 2 mRNAs in transformed rodent cells when compared with their non-transformed counterparts (Cancer Res., 52(1992) 2186-2190). To investigate this phenomenon further the rates of gene transcription and the effects of translation/transcription inhibition on erb A-alpha 2 mRNA expression were examined. The present study found no difference between non-transformed and transformed cells in erb A-alpha 2 gene transcription rate, nor an effect of cycloheximide on erb A-alpha 2 mRNA expression. However, a significant difference was obtained with actinomycin D. With this inhibitor, the half-life of erb A-alpha 2 mRNAs in nontransformed rodent cells was determined to be approximately 8 h. In contrast, the alpha 2 transcripts in their transformed counterparts showed no decay during this period, suggesting that the elevation of erb A-alpha 2 mRNAs in transformed rodent cells was due to increased transcript stability.

Increased messenger RNA levels of the antagonist thyroid hormone receptor erbA-alpha 2 and decreased levels of erbA-alpha 1 and erbA-beta 1 receptor messenger RNAs in neoplastic rodent cells.

Nothern blot analysis of total RNA from the mouse C3H/10T1/2 cell line indicated that the erbA alpha gene transcribed three mRNA species of similar sizes (2.6, 5.5, 6.6 kilobases) as found in rodents. The 2.6-kilobase mRNA (erbA-alpha 2) was approximately 7- to 8-fold more abundant than either the 5.5- (erbA-alpha 1) or 6.6-kilobase species. The expression of the erbA-alpha 2 transcript increased 3- to 30-fold when "normal" mouse or rat cells were growth arrested by concluence. Triiodothyronine, at a concentration of 1 nM, had no effect on the levels of the erbA-alpha mRNA species in confluent cells nor on the levels of erbA-alpha 2 in proliferative normal or transformed C3H/10T1/2 cells. In log-phase growing cells there was a 2.5- to 5-fold increase in the relative expression of erbA-alpha 2 mRNA in transformed mouse C3H/10T1/2 cells, transformed cloned rat embryo fibroblasts (CREF), transformed rat embryo fibroblasts (REF), and a transformed temperature-sensitive rat mutant cell line (ts7E) when compared with their non-transformed counterparts. In contrast to the elevation of erbA-alpha 2 in transformed cells, erbA-alpha 1 and erbA-beta 1 mRNAs decreased in transformed mouse and rat cell lines. In conclusion, it is suggested that the increased levels of the erbA-alpha 2 transcript and the decreased levels of erbA-alpha 1 and erbA-beta 1 in neoplastic cells may account for the loss of thyroid hormone regulation of inducible pathways and decreased nuclear triiodothyronine binding as previously reported.

Cross-linking of G-proteins to the prolactin receptor in rat NB2 lymphoma cells.

In Nb2 cell membranes, two guanine nucleotide-binding protein (G-protein) species (Mr 43.5 and 46.5 kD) were [32P]-ADP-ribosylated by cholera toxin, while a single protein (Mr 41.5 kD) was [32P]-ADP-ribosylated by pertussis toxin. Immunostaining indicated two immunoreactive prolactin (PRL) receptor moieties of 56 and 64 kD. Cross-linking with ethylene glyco bis[succinimidyl-succinate] (mol. length of 16.1 A) generated a high mol. wt., [32P]-ADP-ribosylated band of 140-160 kD which also showed immunoreactivity with antiserum to the PRL receptor. Other cross-linkers with shorter molecular lengths (8.6 - 11.4 A) were ineffective. These findings indicate that the Nb2 lactogen receptor is complexed with G-proteins and provide evidence for the role of G-proteins in mediating PRL-stimulated mitognesis in Nb2 cells.