Heparin-binding growth factor gene expression and receptor characteristics in normal rat prostate and two transplantable rat prostate tumors.

Heparin-binding polypeptide growth factors (HBGF) are essential mitogens for isolated prostate cells. HBGF type one (HBGF-1) mRNA was expressed specifically in the epithelial cells of prostates from normal 6- to 8-week-old rats. Expression declined significantly at 14 weeks and was undetectable in 35-week-old animals. Slow-growing, androgen-responsive, nonmetastatic Dunning R3327PAP tumors, which are composed of a well-defined epithelium and stroma, expressed HBGF-1 mRNA constitutively in specifically the mesenchymal cells. A rapid-growing, androgen-independent, metastatic variant (Dunning R3327AT-3), which was composed of a single clonogenic cell type, expressed both HBGF-1 and HBGF type two (HBGF-2) mRNA. HBGF activity in the extracts of normal and tumor tissues correlated with mRNA levels. Epithelial cells from the R3327PAP tumor and the single cell type that composed the R3327AT-3 tumor exhibited alterations in HBGF receptor characteristics that correlated with increased sensitivity to mitogenic effects of HBGF. The results suggest that alterations in HBGF gene expression in both prostate epithelial and mesenchymal cells and in properties of the receptor in specifically epithelial cells may contribute to differential growth rates and malignancy of different prostatic tumors.

Androgens and glucocorticoids modulate heparin-binding growth factor I mRNA accumulation in DDT1 cells as analyzed by in situ hybridization.

The ductus deferens smooth muscle tumor cell line (DDT1-MF-2) is very sensitive to steroids. Treatment with 10 nM testosterone accelerates the growth of DDT1 cells in the absence of serum. Glucocorticoids in the presence or absence of androgens inhibits growth. Stimulation of growth of DDT1 cells by testosterone can be replaced by the addition of heparin-binding growth factor I and II (HBGF). Addition of testosterone plus HBGF growth factors results in a further increase in cell number. By in situ hybridization, accumulation of HBGF-I mRNA is significantly increased by testosterone treatment of low density cultures. Testosterone treatment of high density cultures results in no stimulation of HBGF-I mRNA accumulation. Glucocorticoids alone, which block growth of DDT1 cells, have no effect of HBGF-I mRNA accumulation. However, the simultaneous addition of glucocorticoid and androgens to DDT1 cells results in a rapid accumulation of HBGF-I mRNA by 12 h, although growth is inhibited by the presence of both steroid analogs.

Double stranded RNA in heterogeneous nuclear RNA from normal and chronic lymphocytic leukemic lymphocytes.

Tritium labelled heterogeneous nuclear RNA (HnRNA) from normal and chronic lymphocytic leukemic (CLL) lymphocytes was investigated before and after fractionation into non-poly(A) containing (-HnRNA) and poly(A) containing (+HnRNA) HnRNA with respect to double stranded RNA (dsRNA). Statistically significant higher amounts of rapidly labelled RNA were recovered from CLL lymphocytes when compared to normal cases. Within the CLL cases a significant linear correlation (r = 0.95) was found between white blood cell counts and the amount of dsRNA in total HnRNA. After fractionation into (-) and (+) HnRNAs the ratios of dsRNAs, expressed as the dsRNA in (-) HnRNA divided by the dsRNA in (+) HnRNA, was lower than the corresponding values in normal cases for all the CLL cases except one. The relationship between (+) HnRNA and the total dsRNA level was different when comparing CLL and normal lymphocytes indicating a RNA processing abnormality.

Purification of major abundance class of poly(A+)-RNA from rat ventral prostate.

Poly(A+)--RNA from rat ventral prostate was isolated using oligo(dT)-cellulose chromatography. 45% of the total poly(A+)--RNA was a single peak at 10S as demonstrated by centrifugation in a 5-20% sucrose gradient containing 1% SDS. By using complementary DNA probes, it was shown that the 10S RNA contained the major abundance class of poly(A+)--RNA. Denaturing agarose-gel analysis revealed 2 major bands in the 10S poly(A+)--RNA preparation approx. 600 NT and 500 NT (NT = nucleotides) long, resp. Double-stranded 32 P-DNAs complementary to light side and heavy side of the 10S poly(A+)--RNA peak were synthesized and isolated using reverse transcriptase and hydroxyapatite (HAP) chromatography. Approx. 40% of the first strand of the cDNAs were converted to double-stranded structures with a Tm of 88 degrees C. HAP purified double-stranded material was 92% resistant to S1 nuclease. the DNA--DNA reannealing profile of double stranded 32 P-cDNA enriched for the 500 NT band gave a Cot 1/2 of approximately 7 X 10(-4) moles X sec X 1(-1) indicating a complexity for this enriched synthetic gene of 500-600 nucleotide pairs (NTP).

Structure of the androgen dependent SVS VI protein as derived from cDNA.

The 11S poly(A+)RNA from rat seminal vesicle was cloned. By hybrid selection of clones reacting to the low molecular weight region of the 11S peak, a clone containing a new seminal vesicle cDNA sequence called SVS VI was isolated. The DNA sequence of two overlapping cDNAs (pSV24 and pSV33) is presented. The sequence of SVS VI was compared to the previously isolated SVS IV and SVS V cDNAS. Dot hybridization showed that SVS VI is androgen responsive after giving testosterone to castrated rats. The hydrophilicity was analyzed using standard Bionet procedures. All three proteins are extremely variable, rich in alpha-helix and very water soluble. The computer predicted hydrophilicity is compared for SVS VI, V and IV. A small region in the 3'-non-coding area of SVS VI has high similarity to a region in SVS IV mRNA.

Cytotoxic effects of kainate ligands on HEK cell lines expressing recombinant kainate receptors.

Exposure of neurons either for prolonged periods of time or to high concentrations of excitatory amino acids (EAA), such as glutamate, results in neuronal death. Kainate also causes cell toxicity through the glutamate receptors. However, it is unclear whether the kainate receptor itself mediates any of the toxic responses. In the present study, HEK cells expressing the GluR6 +/- KA2 receptor subunit(s) were studied for their susceptibility to toxicity through the kainate receptor by kainate ligands. The natural ligand, glutamate, did not result in toxicity to the recombinant cell lines over that observed with the untransfected HEK cells, whereas kainate produced a 2-3-fold increase in LDH in both the HEK/GluR6 (ANOVA, P = 0.0001) and HEK/GluR6 + KA2 (ANOVA, P = 0.0002) cell lines following treatment with various dosages, but did not affect the HEK cells. Similar 2-3-fold increases in LDH activity were detected in both recombinant cell lines following treatment with 100 nM of SYM2081 ((2S,4R)-4-methylglutamic acid), a dose at which agonistic activity is elicited. The rank order potencies for eliciting toxicity are consistent with the previously reported EC50 values (SYM2081 > kainate > > > glutamate). Surprisingly, the kainate antagonist, NBQX, was the most toxic of the compounds tested although it had an affinity for the kainate receptor similar to glutamate. Treatment with as little as 10 nM elicited a dramatic increase in toxicity (6-10-fold) in the recombinant cell lines. At 1 microM, NBQX was significantly more toxic (Fisher PLSD, P < 0.05) than any of the other compounds tested. Thus, it appears that cell toxicity can be mediated via kainate receptor through two independent mechanisms: activation and blockage of the kainate receptor.

Use of a cloned double stranded cDNA coding for a major androgen dependent protein in rat seminal vesicle secretion: the effect of testosterone in gene expression.

The abundant class of poly(A+)RNA [poly(A+)RNA11S] from rat seminal vesicle was used to synthesize ds-cDNA11S. The ds-cDNA11S was inserted and cloned into the Pst I site of pBR-322 using E. coli RR1 as host. Colony filter hybridization and restriction mapping was used to demonstrate that a 620 NTP long insert in a plasmid clone (pSV2) represents the almost full length structural gene coding for a precursor to the seminal vesicle secretion protein IV (SVS IV). The entire insert was sequenced and the coding region was matched with the known amino acid sequence. Most of the signal peptide sequence was derived from the DNA sequence. The insert in pSV2 was labelled and used to study the effect of testosterone on the accumulation of mRNA SVS IV. Administration of testosterone to castrated rats resulted in the induction of mRNA SVS IV from a few molecules per cell to levels of over 100,000 after 96 h of hormone treatment.

Adenylate-rich sequences of heterogeneous nuclear RNA from normal and chronic lymphocytic leukaemia lymphocytes.

Rapidly labelled high molecular weight heterogeneous nuclear RNA (Hn RNA) from normal and chronic lymphocytic leukaemia (CLL) lymphocytes has been fractionated into two classes by chromatography on poly (U) sephasose. The (+) Hn RNA is bound to poly (U) sepharose and contains both a long poly (A) segment (100-200 nucleotides) and shorter (A) rich sequences (approximately 20-30 nucleotides. (-) Hn RNA is not bound to poly (U) sepharose and lacks the longer poly (A) segment but does contain shorter (A) rich sequences. Partial association of the short (A) rich segments with double-stranded regions was found for both classes of Hn RNA but was most pronounced in (-) Hn RNA from high WBC cases of CLL. Both classes of Hn RNA from CLL lymphocytes contain varying but generally higher amounts of the short (A) rich fraction than those in normals. Similar amounts of the longer poly (A) segments were found in (+) Hn RNA from both CLL and normal lymphocytes.

Heterogeneous nuclear rna from lymphocytes of chronic lymphocytic leukaemia: adenylate-rich and double-stranded regions.

Rapidly labelled high molecular weight nuclear RNA from lymphocytes of chronic lymphocytic leukaemia was analysed for ribonuclease-stable adenylate-rich and double-stranded regions. The polyadenylate content corresponds to 0.4-0.5 percent and the content of double-stranded sequences to 2-4 percent of the total nucleotides. Partial association of polyadenylate segments with double-stranded regions was found by comparative analysis of (3H)-adenosine and (3H)-uridine labelled ribonuclease-stable RNA before and after thermal denaturation. Comparison with normal lymphocytes shows lower proportions of polyadenylate-containing RNA binding to poly(U)-Sepharose in leukaemia cells than in normals. Partial degradation of rapidly labelled high molecular weight RNA was found in leukaemia cases with low white cell counts.

Heparin-binding (fibroblast) growth factors type one and two genes are co-expressed in proliferating normal human vascular endothelial and smooth muscle cells in culture.

Complimentary ribonucleic acid (cRNA) probes were used to detect expression of the genes for heparin binding (fibroblast) growth factor type one (HBGF-1) and two (HBGF-2) in cultured endothelial and smooth muscle cells from normal human blood vessels. Hybridization in situ revealed that transcripts for both HBGF-1 and HBGF-2 are expressed in endothelial cells from both umbilical vein and aorta. Relative intensity of radioactive grains suggest that HBGF-1 gene expression may exceed HBGF-2 expression in aortic smooth muscle cells. Collective expression of both HBGF-1 and HBGF-2 in smooth muscle cells may exceed that in endothelial cells.

Effect of cysteine substitutions on the mitogenic activity and stability of recombinant human keratinocyte growth factor.

Human Keratinocyte growth factor (hKGF), a member of the FGF family of growth factors, contains five cysteines at amino acid positions 1, 15, 40, 102, and 106. We expressed five cysteine mutants of hKGF in which the cysteines were cumulatively replaced with alanine or serine, starting with cysteine-1. Recombinant hKGF has an inherently higher mitogenic activity and stability to heat and acid than reported for glycosylated hKGF. Mitogenic activity is increased an additional 2.6 fold by substitution of cysteine-1 with alanine. Mutants with the conserved cysteine substituted at position 40 were more susceptible to heat inactivation than rhKGF, but showed no significant difference in acid inactivation. Cysteine-free rhKGF is mitogenic, demonstrating that neither cysteines nor disulfide bonds are required for mitogenic activity. However, cysteine-free rhKGF does not bind Heparin-Sepharose and is unstable to heat and acid compared to rhKGF, suggesting that the cysteines have a role in maintaining KGF's structure. This information will useful in the development of a more stable and more potent wound healing agent from hKGF.

Seminal vesicle secretion IV gene: allelic difference due to a series of 20-base-pair direct tandem repeats within an intron.

The rat seminal vesicle secretion IV (SVS IV) gene was isolated from a lambda Charon 4A library. The SVS IV gene transcription unit was found to be on one 3.3-kilobase (kb) EcoRI fragment. Restriction mapping and DNA sequence analysis demonstrated that the entire length of the SVS IV transcription unit is 1,930 base pairs (bp) and contains two introns. The 3.3-kb EcoRI fragment contains 144 bp of 5'-flanking region. At -113 bp from the presumed transcription initiation site an interesting structure with perfect dyad symmetry is noted. In another lambda clone, a 3.5-kb EcoRI fragment was isolated that contains the SVS IV gene and was shown to be identical to the 3.3-kb EcoRI fragment except for 180 bp of DNA in the second intron. The extra DNA consists of several (8-10) 20-bp tandem repeats flanked on each side by seven or eight copies of this same 20-bp repeat. Fisher X Sprague-Dawley hybrid rats, which contain both the EcoRI 3.5-kb form and the 3.3-kb form of the SVS IV gene, were crossed with each other. Analysis of the F1 generation demonstrated that the presence or absence of the 180-bp intronic insertion in the SVS IV gene defines an allelic difference. This report also presents the DNA sequence of the transcription unit and flanking regions of the SVS IV gene.

In vitro translation of major abundant class poly(A+)-mRNA from rat seminal vesicle.

Total poly (A)-containing messenger RNA [poly(A)-mRNAT] was isolated from seminal vesicles of adult male rats. By preparative 5%-20% linear sucrose gradient centrifugation in 1% SDS, a single peak was seen in the 11S region [poly(A)-mRNA11S]. Poly (A)-mRNAT and poly(A)-mRNA11S were translated in the wheat germ S-30 in vitro translation system. Laemmli-SDS-polyacrylamide slab gel electrophoresis of translation products of poly(A)-mRNAT revealed three major protein bands at approximately 18,500, 15,000, and 14,000 daltons. In vitro translation of poly(A)-mRNA11S across the 11S peak revealed partial purification of the mRNAs for the three predominant proteins. The lower molecular weight part of the 11S peak enriched for the mRNAs coding for the 15,000 and 14,000 dalton polypeptides, and the higher molecular weight side of the peak enriched for the 18,500 dalton mRNA. The highly abundant class of mRNA from rat seminal vesicle can be purified with one sucrose gradient centrifugation.

Characterization of the hamster DDT-1 cell aFGF/HGBF-I gene and cDNA and its modulation by steroids.

Syrian hamster DDT-1 cells are derived from smooth muscle of the ductus deferens. DDT-1 cell growth is increased by the addition of testosterone (T). Acidic fibroblast growth factor (aFGF) or basic fibroblast growth factor (bFGF) also known as heparin binding growth factor I and II (HBGF-I and HBGF-II) can replace T in the stimulation of growth in these cells. This phenomenon is correlated with testosterone's ability to elevate aFGF/HBGF-I mRNA. The increase steady-state levels of aFGF/HBGF-I mRNA were documented by northern blots and by in situ hybridization. Using a 520 bp human aFGF/HBGF-I cDNA probe, a genomic clone with a 38 kb DNA insert was isolated from a cosmid library. By restriction enzyme analysis and southern hybridization, it was determined that there are three coding exons. DNA sequence analysis showed all of the coding region and 3' noncoding sequences were on this clone. A 5' noncoding exon not in the 38 kb insert is indicated, based on the cDNA sequences and genomic sequences of aFGF/HBGF-I's from hamster DDT-1 cells and several other species. The cDNA for hamster aFGF/HBGF-I was isolated from a DDT-1 lambda gt11 library and sequenced. Comparison of the coding region of aFGF/HBGF-I from four species shows a greater than 90% conservation of amino acid sequence.

High and low affinity binding of heparin-binding growth factor to a 130-kDa receptor correlates with stimulation and inhibition of growth of a differentiated human hepatoma cell.

The differentiated human hepatoblastoma-derived cell line, HepG2, displayed two classes of specific membrane receptors for heparin-binding growth factor type 1 (HBGF-1). Specific membrane receptors were distinguished from nonreceptor heparin-like binding sites. Receptors with an apparent Kd of 9.2 +/- 0.9 pM and present at 15,000 +/- 900/cell correlated with HBGF-1 stimulation of HepG2 growth. Receptors with an apparent Kd of 2 +/- 0.4 nM and present at 180,000 +/- 18,000/cell correlated with inhibition of growth and changes in secretory products. Other hepatoma cell lines exhibited a simple positive mitogenic response to HBGF-1 and a single class of high affinity binding sites. HBGF-1 covalently cross-linked to hepatoma cell surface polypeptides of apparent mean molecular mass of 130 kilodaltons. At 37 degrees C, receptor-bound HBGF-1 was internalized (t 1/2 = 45 min) but not degraded for up to 6 h. The display of receptors decreased with increased cell density and expression of HBGF-1 mRNA and HBGF-1-like activity in the culture medium. Proliferating normal human hepatocytes also exhibited two classes of binding sites with affinities for HBGF-1 and apparent molecular weight similar to HepG2 cells. These results implicate HBGF-1 or homologues in human hepatoma cell growth and normal liver cell regeneration.

Isolation and partial purification of the major abundant class rat seminal vesicle poly(A+)-messenger RNA.

Total poly(A(+))-RNA (poly(A(+))-RNA(tot)) was isolated from rat seminal vesicle and its size distribution determined by 70% formamide 5-25% sucrose density analysis. One major peak was resolved in the 10-13 S region and accounted for approximately 35% of the total poly(A(+))-RNA applied. Preparative 1% SDS, 5-20% linear sucrose density gradients also resolved a single major peak in the 11S region (poly(A(+))(11S). Analysis of poly(A(+))-RNA(tot) and poly(A(+))-RNA(11S) under denaturing conditions on 2% agarose gel electrophoresis demonstrated two major components in both poly(A(+))-RNA populations. Size estimations for these components are 620 and 540 NT respectively. (3)H-cDNA was made to both poly(A(+))-RNA(tot) and poly(A(+))-RNA(11S). Back-hybridization of poly(A(+))-RNA(tot) and poly(A(+))-RNA(11S) to their respective (3)H-cDNA revealed a highly abundant class representing 41% and 85% of the sequences in their respective (3)H-cDNA's. The highly abundant class corresponded to 3-5 sequences present in 30,000-50,000 copies/cell. Invitro translation of poly(A(+))-RNA(11S) resulted in two major polypeptides coded for by the 620 NT long and 540 NT long poly(A(+))-RNA respectively.Images

Heparin-binding growth factor type 1 (acidic fibroblast growth factor): a potential biphasic autocrine and paracrine regulator of hepatocyte regeneration.

Heparin-binding growth factor type 1 (HBGF-1; sometimes termed acidic fibroblast growth factor) is potentially an important factor in liver regeneration. HBGF-1 alone (half-maximal effect at 60 pM) stimulated hepatocyte DNA synthesis and bound to a high-affinity receptor (Kd = 62 pM; 5000 per cell). Epidermal growth factor (EGF) neutralized or masked the mitogenic effect of HBGF-1 concurrent with appearance of low-affinity HBGF-1 binding sites. HBGF-1 reduced the inhibitory effect of transforming growth factor type beta (TGF-beta) on the EGF stimulus. Nanomolar levels of HBGF-1 decreased the EGF stimulus. An increase in hepatic HBGF-1 gene expression after partial hepatectomy precedes increases in expression of the EGF homolog, TGF-alpha, and nonparenchymal-cell-derived TGF-beta in the regenerating liver. Expression of HBGF-1 mRNA occurs in both hepatocytes and nonparenchymal cells and persists for 7 days in liver tissue after partial hepatectomy. HBGF-1 acting through a high-affinity receptor is a candidate for the early autocrine stimulus that drives hepatocyte DNA synthesis prior to or concurrent with the EGF/TGF-alpha stimulus. It may allow hepatocyte proliferation to proceed in the presence of low levels of TGF-beta. An EGF/TGF-alpha-dependent change in HBGF-1 receptor phenotype and increasing levels of nonparenchymal-cell-derived HBGF-1 and TGF-beta may serve to limit hepatocyte proliferation.

(2S,4R)-4-methylglutamic acid (SYM 2081): a selective, high-affinity ligand for kainate receptors.

Glutamic acid activates ionotropic glutamate receptors that mediate excitatory transmission in the central nervous system. The introduction of a methyl group at position 4 of glutamic acid imparts selectivity for kainate receptors, relative to other (N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) ionotropic glutamate receptors. Among the stereoisomers of 4-methylglutamic acid, the potency of the (2S,4R)-isomer (SYM 2081) to inhibit [3H]kainic acid binding to both wild-type (rat forebrain) and recombinant (GluR6) kainate receptors (IC50 values of approximately 32 and 19 nM, respectively) was comparable to that of kainic acid (IC50 values of approximately 13 and 28 nM, respectively). SYM 2081 was approximately 800- and 200-fold less potent as an inhibitor of radioligand binding to wild-type (rat forebrain) alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate receptors, respectively. Preexposure of human embryonic kidney 293 cells stably expressing GluR6 receptors to low concentrations of SYM 2081 (30-300 nM) resulted in a reversible blockade of the rapidly desensitizing currents produced by kainate application. At higher concentrations, SYM 2081 (EC50 of approximately 1 microM) elicited kainate-like, rapidly desensitizing, inward currents. Pretreatment of recombinant GluR6 receptors with concanavalin A both abolished the effect of SYM 2081 to block kainate-induced currents and revealed nondesensitizing currents induced by SYM 2081 alone. The latter observations provide strong support for the hypothesis that SYM 2081 blocks kainate-induced currents through a process of agonist-induced desensitization. SYM 2081 also activated alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor currents in primary cultures of cerebral cortex and, consistent with data obtained by radioligand binding, was approximately 5-fold less potent than kainate (EC50 values of 325 and 70 microM, respectively) in this measure. SYM 2081 is a high-affinity, selective, kainate agonist that may prove useful both as a probe to examine the physiological functions of kainate receptors and as the prototype of a novel class of therapeutic agents.