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M T Gillespie

Publications and source records attributed to M T Gillespie.

At least 55 records · Page 3Linked to original sources
PubMed1998

Parathyroid hormone-related protein (PTHrP) mRNA splicing and parathyroid hormone/PTHrP receptor mRNA expression in human placenta and fetal membranes.

During human pregnancy, parathyroid hormone-related protein (PTHrP) and parathyroid hormone (PTH)/PTHrP receptor are produced by the uterus, placenta, fetal membranes (amnion and chorion) and developing fetus. PTHrP alternative 3' mRNA splicing results in transcripts which encode three PTHrP isoforms and have been identified in amnion. Uteroplacental PTHrP expression is greatest in amnion and increases dramatically during late pregnancy. The aims of this study were to determine PTH/PTHrP receptor mRNA expression at preterm and term gestations and to determine 3' alternative splicing patterns in placenta, amnion and choriodecidua at preterm and term gestations. Using semiquantitative reverse transcription-polymerase chain reaction, PTHrP and PTH/PTHrP receptor transcripts were identified in preterm (n=5) and term (n=7) gestational tissues. PTH/PTHrP receptor mRNA expression did not differ between tissue types or change with advancing gestation. In contrast, PTHrP expression in the same tissues increased with advancing gestation and was significantly greater in amnion than in placenta and choriodecidua. Thus PTHrP, although produced predominantly in amnion, may act in amnion and other tissues including placenta, choriodecidua and myometrium. In amnion over placenta, transcripts encoding PTHrP 1-139 and 1-173 were detected in some preterm and all term samples and those encoding PTHrP 1-141 were detected in all samples. Similar results were obtained for reflected amnion. In placenta and choriodecidua, PTHrP 1-139 and 1-173 transcripts were undetectable or of low abundance. PTHrP 1-141 transcripts were detected in some placenta and choriodecidua samples. In summary, transcripts encoding PTHrP 1-141 appeared to be more abundantly expressed than those encoding PTHrP 1-139 or 1-173. However, the up-regulation of PTHrP expression in amnion at term may involve each of the alternative 3' mRNA splicing pathways since transcripts for each isoform appeared to be more consistently expressed at term.

Alternative Splicing↗
PubMed1997

Calcitonin receptors, bone sialoprotein and osteopontin are expressed in primary breast cancers.

Several human breast cancer cell lines express the calcitonin receptor (CTR), but this has not been demonstrated previously in clinical breast cancers. We examined 18 primary breast cancers by reverse transcription-PCR, for expression of CTR and of the bone proteins osteopontin (OPN) and bone sialoprotein (BSP). OPN and CTR were expressed by each of the tumours, and 7 (39%) additionally expressed an alternate form of CTR, whilst BSP was expressed by 13 tumours (72%). In situ hybridisation confirmed that expression of OPN and CTR was confined to the tumour cells. Expression of CTR, BSP and OPN may prove to be a useful marker for breast cancers, and their role in the homing of breast cancer cells to bone remains to be investigated.

Breast Neoplasms↗
PubMed1997

Frequent loss of heterozygosity in early non-small cell lung cancers at chromosome 9p21 proximal to the CDKN2a gene.

Deletions involving the chromosome 9p21 region have been reported as frequent events in non-small cell lung cancer (NSCLC). To investigate potential tumor-suppressor gene (TSG) loci within the 9p21 region, which also harbors the candidate TSG locus CDKN2a, we studied 32 cases of primary NSCLC for loss of heterozygosity (LOH). Tumor and paired normal lung cells were microdissected from lung tissue imprints and all samples screened using PCR-LOH analysis with 15 9p markers. In addition, 3 NSCLC cell lines and their matched normal lung and tumor DNA were similarly analyzed. LOH at the marker D9S259, which is proximal to the CDKN2a locus, was found most frequently (52%), while LOH at D9S942, the marker closest (5 kb) to the CDKN2a gene, was seen in only 17%. Homozygous loss of markers close to CDKN2a was, however, detected in 2 of the 3 cell lines and one accompanying tumor sample. We propose that a TSG in the region of deletion proximal to the CDKN2a gene within 9p21 may play a significant role in the pathogenesis and progression of NSCLC.

Carcinoma, Non-Small-Cell Lung↗
PubMed1997

Interleukin-18 (interferon-gamma-inducing factor) is produced by osteoblasts and acts via granulocyte/macrophage colony-stimulating factor and not via interferon-gamma to inhibit osteoclast formation.

We have established by differential display polymerase chain reaction of mRNA that interleukin (IL)-18 is expressed by osteoblastic stromal cells. The stromal cell populations used for comparison differed in their ability to promote osteoclast-like multinucleated cell (OCL) formation. mRNA for IL-18 was found to be expressed in greater abundance in lines that were unable to support OCL formation than in supportive cells. Recombinant IL-18 was found to inhibit OCL formation in cocultures of osteoblasts and hemopoietic cells of spleen or bone marrow origin. IL-18 inhibited OCL formation in the presence of osteoclastogenic agents including 1alpha,25-dihydroxyvitamin D3, prostaglandin E2, parathyroid hormone, IL-1, and IL-11. The inhibitory effect of IL-18 was limited to the early phase of the cocultures, which coincides with proliferation of hemopoietic precursors. IL-18 has been reported to induce interferon-gamma (IFN-gamma) and granulocyte/macrophage colony-stimulating factor (GM-CSF) production in T cells, and both agents also inhibit OCL formation in vitro. Neutralizing antibodies to GM-CSF were able to rescue IL-18 inhibition of OCL formation, whereas neutralizing antibodies to IFN-gamma did not. In cocultures with osteoblasts and spleen cells from IFN-gamma receptor type II-deficient mice, IL-18 was found to inhibit OCL formation, indicating that IL-18 acted independently of IFN-gamma production: IFN-gamma had no effect in these cocultures. Additionally, in cocultures in which spleen cells were derived from receptor-deficient mice and osteoblasts were from wild-type mice and vice versa, we identified that the target cells for IFN-gamma inhibition of OCL formation were the hemopoietic cells. The work provides evidence that IL-18 is expressed by osteoblasts and inhibits OCL formation via GM-CSF production and not via IFN-gamma production.

Animals↗
PubMed1997

PTHrP and cell division: expression and localization of PTHrP in a keratinocyte cell line (HaCaT) during the cell cycle.

Parathyroid hormone-related protein (PTHrP) is highly expressed in normal skin keratinocytes, and its involvement in growth and differentiation processes in these cells has been implicated by several lines of evidence which include the use of antisense PTHrP (Kaiser et al., 1994, Mol. Endocrinol., 8:139-147). In this study, we have investigated whether PTHrP expression and its subcellular localization is linked to cell cycle progression in a human keratinocyte cell line (HaCat), which constitutively expresses and secretes PTHrP. PTHrP mRNA and immunoreactive PTHrP were assessed in asynchronous dividing cells and in cells blocked at G1 or G2 + M phases of the cell cycle using several different protocols. The response of PTHrP mRNA expression was examined following readdition of serum in the continued presence of cycle blockers, and after release from cell cycle block, or from cell synchronization by serum deprivation. PTHrP expression was greatest in actively dividing cells when cells were in S and G2 + M phases of the cell cycle and were lowest in quiescent G1 cells. Most notable were the high levels of PTHrP mRNA and protein in cells at G2 + M phase of the cell cycle at division. Furthermore, PTHrP was localized to the nucleolus in quiescent cells, but redistributed to the cytoplasm when cells were actively dividing. Taken together, these results support a role for PTHrP in cell division in keratinocytes. In asynchronously growing cells, PTHrP expression fell as cells became confluent at a time when cell growth is inhibited and cells begin to differentiate. Mitogen stimulation of HaCaT cells resulted in a rapid increase in PTHrP mRNA expression, but was dependent upon cells being in the G1 phase of the cell cycle. Cells blocked in G1 responded to mitogen both in the continued presence of aphidicolin or when released from block. Cells blocked at G2 + M with colcemid expressed high levels of PTHrP mRNA and protein, and PTHrP mRNA did not respond further to mitogen in the continued presence of blocker. However, in cells released from block at G2 + M by addition of serum, an increase in PTHrP expression was seen coincident with the progression of cells into G1. In contrast, in a squamous cancer cell line (COLO16), basal PTHrP expression was high and was not altered during the cell cycle or by cell cycle block, consistent with association of its dysregulated expression in malignant cells. The results of this study suggest that PTHrP may have two roles in the cell cycle; one in G1 in response to mitogen, and a second at cell division when its expression is high and it is relocated from the nucleolus to the cytoplasm.

Aphidicolin↗
PubMed1997

The expression of parathyroid hormone-related protein mRNA and immunoreactive protein in human amnion and choriodecidua is increased at term compared with preterm gestation.

Parathyroid hormone-related protein (PTHrP) gene expression and/or immunoreactive protein have previously been identified in the uterus and intrauterine gestational tissues. The putative roles of PTHrP during pregnancy include vasodilatation, regulation of placental calcium transfer, uterine smooth muscle relaxation and normal fetal development. The aims of this study were 1) to determine the tissue-specific and temporal expression of PTHrP mRNA and immunoreactive protein in human gestational tissues collected at preterm and term; and 2) to determine the effect of labour on PTHrP expression by collecting these tissues from women undergoing elective caesarean section (before labour), intra-partum caesarean section during spontaneous-onset labour (during labour), and women with spontaneous labour and normal vaginal delivery (after labour). Total RNA and protein were extracted from placenta, amnion (over placenta and reflected) and choriodecidua for analysis by Northern blot (using a specific human PTHrP cDNA probe), and by N-terminal PTHrP RIA respectively. In amnion over placenta, reflected amnion and choriodecidua both PTHrP mRNA relative abundance and immunoreactive protein were significantly elevated at term compared with preterm (P < 0.01). At term, both PTHrP and its mRNA were significantly greater in amnion than in placenta and choriodecidua (P < 0.05). Also, both PTHrP and its mRNA were significantly elevated in amnion over placenta compared with reflected amnion (P < 0.05). The expression of PTHrP and its mRNA did not change in association with term labour or rupture of the fetal membranes, therefore this study provides no evidence for a specific PTHrP role in the onset and/or maintenance of term labour. However, the significant up-regulation of PTHrP mRNA and protein in the fetal membranes at term compared with preterm suggests an important role in late human pregnancy.

Amnion↗
PubMed1996

PC8 [corrected], a new member of the convertase family.

A novel subtilisin-like protein, PC8, was identified by PCR using degenerate primers to conserved amino acid residues in the catalytic region of members of the prohormone convertase family. PC8 was predicted to be 785 residues long and was structurally related to the mammalian convertases furin, PACE4, PC1 and PC2, sharing more than 50% amino acid identity over the catalytic region with these family members. PC8 possessed the catalytically important Asp, His, Asn and Ser amino acids, the homo B domain of this family of enzymes and a C-terminal hydrophobic sequence indicative of a transmembrane domain. Structurally, PC8 is more related to furin and PACE4 than to PC1 or PC2. Like furin and PACE4, PC8 mRNA was found to be widely expressed; this is in contrast with PC1 and PC2, which have a restricted distribution. Two transcripts, of 4.5 and 3.5 kb, were detected in both human cell lines and rat tissues. Unlike furin and PACE4, both of which map to chromosome 15, PC8 maps to chromosome 11q23-11q24, suggesting that this gene may have resulted from an ancient gene duplication event from either furin or PACE4, or conversely that these genes arose from PC8.

Amino Acid Sequence↗
PubMed1996

Expression of parathyroid hormone-related protein in cells of osteoblast lineage.

The expression of parathyroid hormone-related protein (PTHrP) was studied in a range of cell cultures representative of the osteoblast lineage and in rat calvarial sections. Primary newborn rat calvarial cells, a rat preosteoblastic cell line (UMR 201), a mouse stromal cell line (ST 2), a mouse calvaria-derived osteoblastic cell line (KS 4), and rat osteosarcoma cell lines (UMR 106-01 and -06), all expressed PTHrP when examined by reverse transcription polymerase chain reaction (RT-PCR). Using a radioimmunoassay we also demonstrated PTHrP in the conditioned medium of the cultured cells, with the exception of UMR 106-01 and -06 cells. Treatment of UMR 201 cells with all-trans-retinoic acid which induces them to acquire a more differentiated phenotype, also led to a time-dependent decrease in PTHrP mRNA levels as determined by RT-PCR, Northern blot analysis, and in situ hybridization. Decreased PTHrP levels in the conditioned medium of the treated cells was also observed. These results suggested that PTHrP production might be greater in less mature osteoblasts. Examination of the populations obtained from newborn rat calvariae by sequential collagenase digestion revealed that the early digests exhibited low ALP activity, low expression of PTH/PTHrP receptor mRNA, and no adenylate cyclase response to PTHrP(1-34). These populations showed the highest level of mRNA and production of PTHrP. Cells from later digests, the "osteoblast-rich" populations, had reduced PTHrP expression. Immunohistochemistry and in situ hybridization in sections of newborn rat calvariae showed PTHrP expression in cuboidal osteoblasts located adjacent to bone and in spindle-shaped cells in the periosteal region. It is concluded that PTHrP is produced by cells of the osteoblast lineage, supporting the hypothesis that PTHrP may function physiologically as a paracrine factor in bone.

Animals↗
PubMed1996

Cell-specific and regulator-induced promoter usage and messenger ribonucleic acid splicing for parathyroid hormone-related protein.

PTH-related protein (PTHrP) is the principle mediator of the syndrome of humoral hypercalcemia of malignancy and has potential paracrine actions on smooth muscle, epithelial cell growth, and placental calcium transport. The human PTHrP gene is complex: a combination of three promoters, one 5' alternative splicing event and alternative 3' splicing, which produces three PTHrP isoforms (139, 141, or 173 amino acids), results in multiple PTHrP messenger RNA (mRNA) species. We employed the RT-PCR technique to identify promoter usage and splicing patterns in a range of human cell lines. Cell line-specific utilization of the promoters and the 3' alternative splicing pathways was detected among bone, breast, kidney, and lung cell lines, although each cell line could potentially produce the three PTHrP isoforms. We also determined whether some of the known regulators of PTHrP differentially modulate promoter usage or splicing patterns. Dexamethasone decreased the abundance of each of the alternative mRNA species. In contrast, epidermal growth factor and transforming growth factor-beta treatment increased the abundance of each PTHrP mRNA species, with particularly marked effects on promoter 1- and promoter 2-initiated transcripts, especially those containing exon VII or VIII. Epidermal growth factor treatment was found to alter PTHrP splicing patterns in a manner consistent with increased transcription from promoters 1 and 2 and stabilization of exon VII- and IX-containing transcripts.

Base Sequence↗
PubMed1995

Modulation of the signal recognition particle 54-kDa subunit (SRP54) in rat preosteoblasts by the extracellular matrix.

Rat preosteoblastic cells, UMR201, develop a more mature phenotype when subcultured onto a type I collagen gel when compared with their growth on plastic. Basal osteopontin mRNA expression is up-regulated, whereas retinoic acid-induced alkaline phosphatase expression is reduced in cells on collagen when compared with cells plated onto plastic. We have used differential display polymerase chain reaction (PCR) of mRNA to identify other mRNA species that are regulated by collagen and/or retinoic acid in UMR201 cells. A number of differentially expressed PCR products were isolated, whose sequences did not correspond to known sequences in the data bank. However, one species which was up-regulated by growth on collagen showed 95 and 94% homology to the murine and canine 54-kDa subunit of the signal recognition particle (SRP54), respectively. In time course experiments, using reverse transcription PCR, it was found that SRP54 mRNA was up-regulated in UMR201 cells as early as 1 h after subculture onto collagen, when compared with cells subcultured onto plastic, and levels remained elevated after 48 h. The increased expression of SRP54 paralleled the increased expression of a known secreted protein, osteopontin. SRP54 recognizes signal sequences of proteins destined for secretion and retards them for further elongation in the endoplasmic reticulum. The increased expression may correlate with the synthesis of specific extracellular matrix molecules in differentiating osteoblasts.

Amino Acid Sequence↗
PubMed1995

Epidermal growth factor-stimulated parathyroid hormone-related protein expression involves increased gene transcription and mRNA stability.

Epidermal growth factor (EGF) produced rapid and striking effects on parathyroid hormone-related protein (PTHrP) gene expression in the immortalized human keratinocyte cell line, HaCaT. Steady-state levels of PTHrP mRNA and secreted PTHrP were increased 10-fold by maximally effective concentrations of EGF. EGF increased both PTHrP gene transcription and PTHrP mRNA stability. Nuclear run-on assays demonstrated a 4-fold increase in transcriptional rate in EGF-stimulated cells while transient transfection analysis indicated that the action of EGF on transcription involved both the GC-rich promoter, P2, and the downstream TATA promoter, P3, but apparently not the upstream TATA promoter, P1. In experiments where EGF treatment produced more stable PTHrP transcripts, the half-life of c-fos mRNA was unaltered, suggesting a relatively specific effect of EGF. Moreover, only those species of PTHrP mRNA containing two of the alternative 3' exons (exons VII and VIII) were stable, those containing exon IX were not. Reverse-transcription PCR demonstrated that EGF produced differential increases in the abundance of PTHrP mRNA species initiated by the three PTHrP promoters. The major effect was seen on the abundance of transcripts initiated by P1 and P2, with less marked regulation of P3-initiated transcripts. Thus EGF regulation of PTHrP gene expression in HaCaT cells is multifactorial and the combination of its actions at the 5' and 3' ends of the gene favours the accumulation of subpopulations of PTHrP mRNA containing exons I, VII and VIII.

Cell Line, Transformed↗
PubMed1995

Evidence for the synthesis of parathyroid hormone-related protein (PTHrP) by nontransformed clonal rat osteoblastic cells in vitro.

Parathyroid hormone-related protein (PTHrP) is synthesized by a variety of tumors and is thought to be the main cause of the clinical syndrome of humoral hypercalcemia of malignancy (HHM). In addition to its parathyroid hormone (PTH)-like actions, novel actions of PTHrP on placental calcium transport and inhibition of in vitro osteoclast activity have been demonstrated. The fact that osteoblasts act as mediators of osteoclastic bone resorption prompted us to investigate whether nontranformed, osteoblastlike cells produce PTHrP. PTHrP has been detected in developing human fetal bones and in rat long bones in culture. For this study, osteogenic cells, CRP 5/4 and CRP 10/30, were employed. Both cell types represent clonal bone cell populations established from 1-day-old rats. While CRP 10/30 cells express the osteoblastic phenotype, CRP 5/4 cells resemble cells with preosteoblastic properties. With a radioimmunoassay (RIA), utilizing antiserum directed against the amino-terminal PTHrP(1-40), it was found that both cell types synthesize PTHrP constitutively. CRP 10/30 cells produce about twice as much as CRP 5/4 cells. Transforming growth factor-beta (TGF-beta 1) was shown to increase the synthesis of PTHrP in CRP 5/4 cells by about 2.5-fold, while in CRP 10/30 cells it caused an approximate 50% reduction of PTHrP. Employing the reverse transcriptase polymerase chain reaction (RT-PCR) technique it was found that both bone cell types express mRNA for PTHrP and that the modulation of the PTHrP mRNA levels by TGF-beta 1 in CRP 5/4, and to a lesser degree in CRP 10/30 cells, was reflected in a change in the level of PTHrP protein in the culture medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Alternative promoter usage and mRNA splicing pathways for parathyroid hormone-related protein in normal tissues and tumours.

The parathyroid hormone-related protein (PTHrP) gene consists of nine exons and allows the production of multiple PTHrP mRNA species via the use of three promoters and 5' and 3' alternative splicing; as a result of 3' alternative splicing one of three protein isoforms may be produced. This organisation has potential for tissue-specific splicing patterns. We examined PTHrP mRNA expression and splicing patterns in a series of tumours and normal tissues, using the sensitive reverse transcription-polymerase chain reaction (RT-PCR) technique. Use of promoter 3 and mRNA specifying the 141 amino acid PTHrP isoform were detected in all samples. Transcripts encoding the 139 amino acid isoform were detected in all but two samples. Use of promoters 1 and 2 was less widespread as was detection of mRNA encoding the 173 amino acid isoform. While different PTHrP splicing patterns were observed between tumours, no tissue- or tumour-specific transcripts were detected. In comparing normal and tumour tissue from the same patient, an increase in the number of promoters utilised was observed in the tumour tissue. Furthermore, mRNA for the PTH/PTHrP receptor was detected in all samples, thus the PTHrP produced by these tumours may potentially act in an autocrine or paracrine fashion.

Base Sequence↗
PubMed1995

Parathyroid hormone-related protein.

We review the current state of knowledge of the molecular properties and actions of parathyroid hormone-related protein (PTHrP) both in cancer patients and in normal physiology. PTHrP is a common product of squamous cancers and is the major mediator of the syndrome of humoral hypercalcemia of malignancy (HHM) by its actions through parathyroid hormone receptors in bone and kidney. Recently developed radioimmunoassays and tissue localization techniques indicate that PTHrP is produced by many more cancers than was originally indicated by clinical studies and that it contributes significantly to malignancy-related hypercalcemia associated with other etiologies, for example, cancers metastatic to bone and hematological malignancies. The gene encoding PTHrP is complex, with multiple exons coding for up to 12 alternate transcripts and three different length proteins, potentially in a tissue-specific manner, by the use of three promoters. Its expression is regulated by hormones and growth factors, and the untranslated exons display features in common with many cytokine genes. Although potential endocrine actions of PTHrP are evident in fetal development, further evidence suggesting that the normal physiological role of PTHrP is predominantly as a locally produced regulator/cytokine comes from localization studies and investigations of its actions in a variety of tissues. Such studies indicate that in addition to its parathyroid hormone-like actions, PTHrP has multiple activities, including those in fetal development, placental calcium transfer, lactation, smooth muscle relaxation, and on epithelial cell growth. Although PTHrP was discovered because of its production by cancers, evidence for its actions as a local regulator highlights the importance of understanding its roles not only in the etiology of HHM in cancer patients but also in normal tissues.

Amino Acid Sequence↗
PubMed1994

Homozygous and hemizygous deletions of 9p centromeric to the interferon genes in lung cancer.

Cytogenetic analysis has indicated that deletion of chromosome 9p occurs in a significant number of non-small cell lung and mesothelioma tumors. Using paired oligonucleotide primers, we have undertaken an extensive analysis of 9p markers to determine homozygous and heterozygous loss of marker sequences. Homozygous loss of D9S169 and D9S171, both of which map centromeric to the IFN gene cluster, were noted in three cell lines (27%) and hemizygous deletions of one or both of these loci was found in a further six cell lines (54%). These data suggest the presence of a potential tumor suppressor gene for lung cancer in proximity to D9S169 and D9S171 at 9p21.

Carcinoma, Non-Small-Cell Lung↗
PubMed1994

Characterisation of sin, a potential recombinase-encoding gene from Staphylococcus aureus.

The staphylococcal beta-lactamase (Bla) transposon Tn4002 has previously been reported to have a high level of insertional specificity for a 1.8-kb region on the plasmid pSK1. Nucleotide sequences of this region and of a related region on plasmid pI9789 were determined. Sequence analysis revealed that these two plasmids contain sin, a gene whose deduced product shows similarity to a family of DNA recombinases. Southern hybridisation analysis indicated that sin is located on alpha-, beta- and gamma-families of Bla plasmids and on pSK1 family plasmids. A region of dyad symmetry located upstream from sin on pSK1 and pI9789 was identified as the site of insertions of Tn552 and Tn4002 in separate isolates.

Amino Acid Sequence↗
PubMed1994

Parathyroid hormone-related protein and human papillomavirus in gynecological tumors.

The presence of parathyroid hormone-related protein (PTHrP) and human papillomavirus (HPV) in a series of gynecological tumors from 131 unselected patients was examined. PTHrP was localized immunohistochemically using a highly specific rabbit polyclonal anti-serum against PTHrP(1-16). The results confirmed that gynecological malignancies, although rarely associated with humoral hypercalcemia of malignancy (HHM), stained for PTHrP in a majority of the squamous-cell carcinomas (SCC) at all sites, but only in a minority of adenocarcinomas, and then in areas of squamous metaplasia. This included a series of endometrial tumors. Detection of HPV types was achieved using a polymerase-chain-reaction (PCR) detection system enabling the detection of HPV types 6, 11, 16, 18, 31, 33 and 45. PTHrP production was not directly related to HPV infection, but correlated with the type of tumor.

Adenocarcinoma↗