Squamous cell carcinomas often produce more than a single bone resorption-stimulating factor: role of interleukin-1 alpha*

Several cultured human squamous cell carcinoma cell lines (SCC-4, SCC-12B2, SCC-12F2, EC-GI-10, and BEN) and one normal keratinocyte line (Epy-1) were investigated for the production of bone resorption-stimulating activity (BRSA). Conditioned medium (CM) from each of the six cell lines stimulated bone resorption in neonatal mouse calvariae in culture. The BRSA of SCC-12F2 and EC-GI-10 was inhibited completely by antibody to interleukin-1 alpha (IL-1 alpha), whereas the BRSA in CM from the BEN, SCC-4, SCC-12B2, and Epy-1 cell lines was only partially inhibited by anti-IL-1 alpha. Addition of indomethacin to the calvarial cultures also partially inhibited the BRSA from EC-GI-10, SCC-4, SCC-12B2, and Epy-1 cells; the BRSA from BEN and SCC-12F2 cells was inhibited completely by indomethacin. cAMP production by calvariae was determined after a 60-min incubation with CM. CM from EC-GI-10, BEN, SCC-4, and Epy-1 stimulated cAMP production by bone. Preincubation of CM from BEN, EC-GI-10, SCC-4, and Epy-1 cells with two antisera against PTH-related protein [PTHrP; one specific for two PTHrP-(1-141), the other recognizing both PTHrP-(1-40) and PTHrP-(1-141)] completely inhibited the cAMP-stimulating activity. Using specific enzyme-linked immunosorbent assays for IL-1 alpha and IL-1 beta, IL-1 alpha was measured in CM of the SCC-4, SCC-12B2, SCC-12F2, and Epy-1 cell lines. IL-1 beta was undetectable (less than 0.1 ng/ml) in CM from all cell lines. Our findings indicate that the BRSA secreted by SCC-12F2 cells can be accounted for largely or entirely by IL-1 alpha, while the activity produced by SCC-12B2 includes IL-1 alpha and another unknown factor(s). The BRSA produced by EC-GI-10, BEN, SCC-4, and Epy-1 cells includes both IL-1 alpha and PTHrP. We conclude that IL-1 alpha may be a more prevalent and biologically significant component of the BRSA produced by SCCs than previously recognized.     

Production of interleukin-1 alpha and a parathyroid hormone-like factor by a squamous cell carcinoma of the esophagus (EC-GI) derived from a patient with hypercalcemia

To determine the pathogenesis of humoral hypercalcemia associated with esophageal carcinoma in a 65-yr-old patient, clonal cell lines (EC-GI) were established from the tumor. The EC-GI cells produced bone-resorbing activity which eluted from a Sephadex G-75 column in a broad peak, with an apparent mol wt of 10,000-50,000. In addition to PTH-like factor(s), the EC-GI cells produced a factor with thymocyte proliferation-stimulating activity which had an apparent mol wt of 15,000-20,000. This interleukin-1 (IL-1)-like factor(s) with acidic pI (4.8 and 5.2) exactly coeluted with the bone-resorbing activity upon DEAE-Sepharose ion exchange chromatography. Both bone-resorbing and thymocyte proliferation-stimulating activities were completely inhibited by anti-IL-1 alpha antiserum, but not by anti-IL-1 beta antiserum. Northern blot hybridization studies revealed that EC-GI cells produced exclusively mRNA for IL-1 alpha. Furthermore, fractions containing IL-1-like activity and PTH-like activity synergistically stimulated bone resorption in vitro, and transplantation of EC-GI cells into nude mice caused hypercalcemia in vivo. These findings suggest that IL-1 alpha and PTH-like factor produced by this squamous cell carcinoma synergistically stimulate bone resorption and are related to humoral hypercalcemia in tumor-bearing nude mice and in the patient.     

EGF receptor antiserum inhibits bone resorbing activity produced by a rat Leydig cell tumor associated with the humoral hypercalcemia of malignancy

The humoral hypercalcemia of malignancy (HHM) is a syndrome caused by tumor cells releasing unknown circulating factors which stimulate osteoclastic bone resorption. In the D6 variant of the rat Leydig cell tumor model of HHM, we found that tumor extracts and tumor cell conditioned medium contained a macromolecular bone resorbing factor which coeluted on column chromatography with transforming growth factor activity (TGF). This observation led to the hypothesis that the tumor-derived bone resorbing factor was a TGF which interacts with the epidermal growth factor (EGF) receptor. To test this hypothesis, we examined the effects of two classes of antisera to the EGF receptor on bone resorption stimulated by conditioned medium from Leydig D6 tumor cells using organ cultures of fetal rat long bones. The antiserum which blocks the binding of EGF to its receptor inhibited bone resorption stimulated by tumor conditioned medium and by EGF. The second antiserum to the EGF receptor which does not block EGF binding or biological activity had no effect on bone resorption stimulated by either tumor conditioned medium or EGF. Neither antiserum had any effect on bone resorption stimulated by parathyroid hormone (PTH). These results indicate that the tumor-derived bone resorbing factor is dependent upon the availability of EGF receptors for its activity and are consistent with it being a TGF.     

Two species of adenylate cyclase-stimulating activity in a murine squamous carcinoma model of humoral hypercalcemia of malignancy

PTH receptor-stimulating proteins may be a common mediator of humoral hypercalcemia of malignancy (HHM). Such proteins exhibit adenylate cyclase-stimulating activity (ACSA) in PTH-sensitive assays, and ACSA has been used to follow their purification. Acid/urea tumor extracts from a murine squamous carcinoma model of HHM were previously shown to have very high ACSA, which was partially, but incompletely, inhibited by the PTH antagonist Nle8,18,Tyr34-bovine PTH-(3-34) amide. ACSA from murine tumor extracts has now been further purified using solvent fractionation and reverse phase HPLC. Approximately half of the ACSA is attributable to a family of three proteins (peaks IA, IB, and IC) with properties characteristic of the PTH receptor-stimulating protein extracted from rat Leydig cell and human HHM tumors. The ACSA in these three peaks of murine tumor extract elutes in the same region as human tumor ACSA on reverse phase HPLC, has a dose-response curve parallel to that of PTH, and is fully inhibited by the PTH-(3-34) antagonist in both the renal cortical and rat osteosarcoma (ROS) adenylate cyclase assays. The remaining half of the ACSA from murine tumor extracts elutes as a single peak (peak II) at a higher acetonitrile concentration on reverse phase HPLC. In the renal cortical assay, its dose-response curve differs from that of PTH, its ACSA is not affected by the PTH-(3-34) antagonist, and it potentiates PTH- or peak I-stimulated adenylate cyclase activity. In the PTH-sensitive intact cell ROS assay, peak II exhibits no ACSA. We conclude that the potent ACSA of murine tumor acid/urea extract results in large part from amplification of the PTH-specific ACSA (peak I) by peak II. Peak II is a distinct protein, not previously reported in tumor extracts, that may act as a postreceptor step in the adenylate cyclase system.     

A tumour necrosis factor alpha polymorphism is not associated with rheumatoid arthritis.

OBJECTIVE--To determine whether a polymorphism within the tumour necrosis factor alpha (TNF alpha) gene is associated with susceptibility to, or severity of, rheumatoid arthritis (RA). METHODS--Consecutive patients with recent onset RA were enrolled in a prospective trial. DNA was collected, disease activity was measured at presentation, and radiographic progression at three years was assessed. Typing of TNF alpha was by polymerase chain reaction and single stranded conformation polymorphism analysis. RESULTS--No association of TNF alpha alleles and susceptibility to, or severity of, RA was demonstrated. CONCLUSIONS--These results indicate that this TNF alpha polymorphism does not play a part in the genetic background of RA.     

A paraneoplastic syndrome of hypercalcemia and leukocytosis associated with solid tumors: production of interleukin 1 alpha (IL-1 alpha) and granulocyte colony-stimulating factor (G-CSF) by clonal squamous cell carcinoma cells

We have demonstrated that T3M-1 cells and T3M-5 cells, derived from squamous carcinomas of patients with leukocytosis and hypercalcemia, produced excessively G-CSF, IL-1 alpha and a small amount of PTH-like factor. We confirmed that G-CSF and IL-1 alpha (hemopoietin 1) enhanced granulocytopoiesis and caused marked leukocytosis in vivo. Furthermore, we also demonstrated that PTH-rP and IL-1 alpha (a potent osteoclast-activating factor) synergistically stimulated bone resorption in vitro and also synergistically increased serum calcium concentration in vivo. Since we have demonstrated that at least two clonal cell lines derived from patients with hypercalcemia and leukocytosis produced G-CSF and IL-1 alpha, we presume that hypercalcemia and leukocytosis associated with some solid tumors may constitute a new paraneoplastic syndrome.     

Co-purification of transforming growth factor beta-like activity with PTH-like and bone-resorbing activities from a tumor associated with humoral hypercalcemia of malignancy

Humoral hypercalcemia of malignancy (HHM) is caused by a circulating bone-resorbing factor or factors. Suggestions as to the nature of this factor include PTH-like proteins, transforming growth factors, and bone-resorbing factors distinct from either of the first two classes of polypeptides. We investigated the occurrence of these three activities in a highly purified extract of the H-500 Leydig cell tumor which causes HHM when implanted into Fisher rats. PTH-like adenylate cyclase-stimulating activity (ACSA) was extracted from tumor tissue by sequential treatment with urea/HCl and ethanol/NaCl. Tumor extract was further purified by hydrophobic-interaction, gel-filtration, and reverse-phase HPLC steps to a specific activity of 1038 ng eq bPTH(1-34)/mg protein. Only the fraction pool containing ACSA demonstrated significant bone-resorbing (1.78-fold over basal) and transforming growth factor activity (epidermal growth factor (EGF)-dependent colony formation in soft agar suspension by NRK-49F indicator cells). A subsequent reverse-phase HPLC step produced material which contained both ACSA and transforming growth factor beta (TGF beta)-like activity in a single fraction. Whether the responsible mediator in this animal model has TGF beta-like properties as well as PTH-like and bone-resorbing activity remains to be determined.     

Increased serum alpha 1-antitrypsin does not produce a rise in fecal alpha 1-antitrypsin in patients with healthy intestines

Fecal alpha-1-Antitrypsin (alpha-1-AT) was related to serum alpha-1-AT concentration in 50 healthy controls, 51 patients with extraintestinal inflammatory disease as well as in 23 patients with ulcerative colitis with mild or severe disease activity. alpha-1-AT concentrations were measured by radial immunodiffusion. Even though serum alpha-1-AT concentration was elevated above normal limits in all patients, a concomitant increase of fecal alpha-1-AT was found only in patients with ulcerative colitis. In these patients a positive correlation between fecal alpha-1-AT-clearance and -concentration and severity and extension of inflammation could be demonstrated. It is concluded that elevated blood levels of alpha-1-AT do not cause increases in fecal alpha-1-AT loss and that fecal alpha-1-AT excretion is increased specifically in patients with intestinal inflammation.     

Epithelial lesions suggestive of a condylomatous origin found closely associated with invasive bronchial squamous cell carcinomas

The histology of the bronchial specimens derived from 104 patients with an established invasive bronchial squamous cell carcinoma was assessed with special reference to the detection of the morphological manifestations of human papilloma virus (HPV) recently described in the uterine cervix, i.e., three different types of condylomatous lesions (papillomatous, flat and endophytic). Histological lesions fulfilling the criteria of papillomatous condyloma were found in 6 cases, those of endophytic condyloma in 4 cases, and lesions identical to flat type condyloma of the uterine cervix were encountered in 26 cases (25%). The role of HPV in the development of these lesions and their relationship to squamous cell carcinogenesis was discussed, and a concept hypothesizing the development of bronchial squamous cell carcinoma is outlined.     

Loss of heterozygosity at chromosome 14q is associated with poor prognosis in head and neck squamous cell carcinomas

Purpose and methods  Loss of heterozygosity (LOH) in a chromosomal location indicates the presence of an inactivated tumor suppressor gene (TSG). Inactivation of TSG has a functional role in the tumorigenesis of head and neck squamous cell carcinoma (HNSCC). Based on the recent evidences of a putative TSG on chromosome 14, we examined LOH on chromosome 14q using eight polymorphic microsatellite markers in 50 cases of HNSCCs. Results  Three regions were detected to have a high LOH rate which included 14q21.2-22.3 (42.5%), 14q31 (55%), and 14q32.1 (37%). The correlation between LOH and clinicopathological findings was investigated through statistical analyses. A strong correlation was observed between the highest LOH marker and the overall and disease-free survival. Conclusions  The results suggest that the distal part of chromosome 14 may host a TSG that may lead to the development and/or progression of HNSCCs. Several genes such as CHES1, BMP4, SAV, and PNN have arisen as candidate tumor suppressors in the region. Davut Pehlivan and Esra Gunduz contributed equally to this work.     

Beta 2-microglobulin is not a bone cell mitogen

During the purification of skeletal growth factor/insulin-like growth factor-II and transforming growth factor-beta (TGF beta) from EDTA extracts of bovine bone matrix significant mitogenic activity for cultured osteoblast (Ob)-like cells eluted in fractions that contained a nearly homogeneous peptide with a mol wt of about 14,000. This peptide has been purified to apparent homogeneity and identified as bovine beta 2-microglobulin (beta 2M) by amino-terminal amino acid sequencing. During the final purification of beta 2M by CN reverse phase HPLC the mitogenic activity for bone cells separated from the beta 2M protein peak. In spite of this the apparently homogeneous beta 2M preparation retained some mitogenic activity. The ED50 of the bone-derived beta 2M (4,890 +/- 462 ng/ml) was several orders of magnitude (2 x 10(3) to 1.4 x 10(5) times) greater than the ED50 of simultaneously assayed purified growth factors and was no different from the ED50 of the crude EDTA matrix extract (3,350 +/- 890 ng/ml). The beta 2M accounted for less than 0.002% of the total mitogenic activity for Ob-like cells present in the extracted matrix proteins. The following lines of evidence suggest that the mitogenic activity of bone matrix-derived beta 2M (BMD-beta 2M) is due to contamination of the BMD-beta 2M with TGF beta rather than an intrinsic property of beta 2M: 1) the coelution of TGF beta through four successive purification procedures and purification of TGF beta from adjoining fractions from C4 reverse phase HPLC; 2) the abolition of biological activity of BMD-beta 2M and TGF beta with reducing agents; 3) the lack of additive stimulation of [3H] methylthymidine incorporation into bone cells when beta 2M was added to maximally active concentrations of purified TGF beta; 4) the reduction of mitogenic activity when the BMD-beta 2M was incubated with anti-TGF beta; and 5) the inhibition of mink lung epithelial proliferation by the beta 2M preparation. Based on these findings we conclude that although beta 2M is present in bovine bone matrix extracts, it is not a mitogen for Ob-like cells.     

Interleukin-3 and interleukin-1 alpha allow earlier bone marrow progenitors to respond to human colony-stimulating factor 1

By using human bone marrow cells enriched for early progenitors by selective immunoadsorption and plated at low cell density (10(3) to 10(4) cells/mL/9.6 cm2) in semisolid methylcellulose culture, we have analyzed the cooperative effects of human colony-stimulating factor 1 (CSF-1), granulocyte-macrophage-CSF (GM-CSF), interleukin-1 alpha (IL-1 alpha), and gibbon as well as human recombinant IL-3 on the formation of monocytic colonies. CSF-1 alone stimulated mature monocytic colony formation by human CFU-M. However, in the presence of IL-3 and erythropoietin, CSF-1 stimulated maximal immature monocytic colony formation at low concentrations and inhibited the formation of granulomonocytic, erythrocytic, and mixed colonies. Cultures with CSF-1 and IL-3 contained more immature monocytic colonies than did cultures with CSF-1 alone. IL-1 alpha alone had little effect. However, IL-1 alpha in combination with optimal concentrations of either CSF-1, GM- CSF, or IL-3 increased the number of colonies containing immature or mature monocytic colonies.     

Lactoferrin is a potent regulator of bone cell activity and increases bone formation in vivo

Lactoferrin is an iron-binding glycoprotein present in epithelial secretions, such as milk, and in the secondary granules of neutrophils. We found it to be present in fractions of milk protein that stimulated osteoblast growth, so we assessed its effects on bone cell function. Lactoferrin produced large, dose-related increases in thymidine incorporation in primary or cell line cultures of human or rat osteoblast-like cells, at physiological concentrations (1-100 microg/ml). Maximal stimulation was 5-fold above control. Lactoferrin also increased osteoblast differentiation and reduced osteoblast apoptosis by up to 50-70%. Similarly, lactoferrin stimulated proliferation of primary chondrocytes. Purified, recombinant, human, or bovine lactoferrins had similar potencies. In mouse bone marrow cultures, osteoclastogenesis was dose-dependently decreased and was completely arrested by lactoferrin, 100 microg/ml, associated with decreased expression of receptor activator of nuclear factor-kappaB ligand. In contrast, lactoferrin had no effect on bone resorption by isolated mature osteoclasts. Lactoferrin was administered over calvariae of adult mice for 5 d. New bone formation, assessed using fluorochrome labels, was increased 4-fold by a 4-mg dose of lactoferrin. Thus, lactoferrin has powerful anabolic, differentiating, and antiapoptotic effects on osteoblasts and inhibits osteoclastogenesis. Lactoferrin is a potential therapeutic target in bone disorders such as osteoporosis and is possibly an important physiological regulator of bone growth.     

Enhancement of cyclic adenosine monophosphate content in bone cells by the factor extracted from a pancreatic cancer associated with hypercalcemia

A woman with exocrine pancreatic cancer presented a syndrome of humoral hypercalcemia of malignancy (HHM). Either urea extract or acid/ethanol extract of the tumor showed a dose-dependent activity to elevate cyclic adenosine monophosphate (AMP) level in rat bone cells in primary culture. When each population obtained by the sequential digestion of rat fetal calvaria was cultured individually and cyclic AMP responses to parathyroid hormone (PTH), calcitonin, and tumor extract were examined, tumor extract-sensitive cells showed a similar distribution to PTH-sensitive cells. Tumor extract and PTH, but not calcitonin, increased cyclic AMP in osteogenic cell line MC 3T3-E1. PTH receptor-mediated increase of cyclic AMP was indicated by an antagonistic action of PTH analogue, (3-34) hPTH, on increase of cyclic AMP in MC 3T3-E1 elicited by tumor extract. Human breast cancer derived cell line MCF-7 had calcitonin-sensitive adenylate cyclase, but neither PTH nor tumor extract increased cyclic AMP in the cells. On Bio-Gel P-60 column, the activity to stimulate bone cell cyclic AMP was eluted as a single peak at the molecular size between 6.5 K and 12.4 K. It was concluded that pancreatic cancer, although rather exceptional as a cause of HHM, produced a factor very similar to that reported in representative HHM tumors of human and animal models.     

Synthetic and partially-purified adenylate cyclase-stimulating proteins from tumors associated with humoral hypercalcemia of malignancy inhibit phosphate transport in a PTH-responsive renal cell line

Hypophosphatemia and hyperphosphaturia characteristically occur in patients with humoral hypercalcemia of malignancy (HHM). To determine if a tumor product causes these abnormalities in phosphate metabolism, rather than, for example, hypercalcemia, we investigated the effect of partially-purified adenylate cyclase-stimulating activity (ACSA) from human and animal HHM-associated tumors on sodium-dependent phosphate transport (Na PiT) in a PTH-responsive renal epithelial cell line. Thirty minute exposure to 7 X 10(-10) MbPTH (1-34) equivalents of ACSA from the human and animal tumors, reduced NaPiT by 20% and 14%, respectively. We also recently isolated an adenylate cyclase-stimulating protein (hACSP) from two human tumors associated with HHM and identified a cDNA clone for this protein which encodes a 141 amino-acid peptide. Based on the deduced amino-acid sequence, we synthesized tyr36 (1-36) hACSP. This synthetic peptide induced a 22% decrease in the initial rate of NaPiT by the epithelial monolayer. Its inhibitory activity was roughly equipotent to that of bPTH (1-34). We conclude that the ACSP derived from HHM-associated tumors decreases phosphate transport in renal epithelial cells. This peptide appears to play a key role in mediating the changes in phosphate metabolism in this syndrome.