Cellular localization of PDGF mRNAs in developing human forebrain.

Platelet-derived growth factor (PDGF) has been implicated in the processes regulating gliogenesis in the CNS. Conflicting in vivo data in rodents have variously implicated either glia or neurons as being the primary source of PDGF. We have used in situ hybridization and immunocytochemical analysis to study the in vivo expression and cellular localization of PDGF-A, sis/PDGF-B, together with the two PDGF receptors alpha and beta, in developing human forebrain. In this study we demonstrate the strong expression of mRNA and protein of both PDGF chains, A and B, and their receptors, alpha and beta, in human embryonic glial cells. The neurons, in contrast to glial cells, expressed lower levels of PDGF and PDGF-receptor mRNAs and protein. Identification of the cell types expressing the PDGF and PDGF-receptor mRNAs was achieved by counterstaining with antibodies specific for glial cells (GFAP) and neurons (NF). The predominant glial-specific expression of both PDGF-A and PDGF-B, together with the coexpression of their receptors alpha and beta, suggests an important role for the PDGF isoforms in the development of human embryonic glial cells and neurons in vivo.

Retroviral vector-modified bone marrow stromal cells secrete biologically active factor IX in vitro and transiently deliver therapeutic levels of human factor IX to the plasma of dogs after reinfusion.

Canine bone marrow stromal cells (BMSCs), transduced ex vivo with retroviral vectors, expressed and secreted biologically active human and canine coagulation factor IX (hFIX and cFIX) in vitro, and on autologous reinfusion expressed hFIX into the circulation of normal (nonhemophiliac) dogs. Human FIX, when expressed in vitro by BMSCs of two dogs at 1.22 and 1.39 microg/10(6) cells/24 hr in medium supplemented with vitamin K, respectively, exhibited 28.1 and 27.3% normal biological activity as determined on the basis of a one-stage clotting assay. BMSCs of two additional dogs expressed 1.54 and 4.81 microg of cFIX/10(6) cells/24 hr in vitamin K-supplemented medium and the expressed cFIX possessed 58.4 and 32.9% normal activity, respectively. Between 2.33 and 3.35 x 10(8) transduced BMSCs, expressing 1.22 and 2.61 microg of hFIX/10(6) cells/24 hr or 3.24 and 7.82 microg of cFIX/10(6) cells/24 hr were reintroduced into the four donor dogs by intravenous infusion. Human FIX was detected in plasma for 7 or 12 days after BMSC reinfusion, with peak levels of 85.8 and 233.0 ng/ml observed at 2 days. Canine anti-hFIX antibodies, which were detected as early as 2-4 days after reinfusion of BMSCs expressing hFIX, may have masked potentially longer duration expression in vivo. Peak plasma levels of hFIX represented 2.1 and 5.8% normal human hFIX levels. When adjusted for percent normal one-stage clotting activity determined in vitro, these levels represented 0.6 and 1.6% normal human hFIX activity levels. Thus, we have demonstrated that retroviral vector-modified BMSCs can deliver human therapeutic levels of hFIX to the circulation of dogs.

Familial meningioma: analysis of expression of neurofibromatosis 2 protein Merlin. Report of two cases.

Meningiomas are primarily benign brain tumors thought to arise through multistep tumorigenesis, involving both the activation of oncogenes and the loss of tumor suppressor genes. The recently isolated neurofibromatosis 2 (NF2) tumor suppressor gene has been found to be mutated in a large proportion of meningiomas. Almost all cases of familial meningioma occur in association with NF2. Familial meningioma in isolation from NF2 (sporadic) is exceedingly rare, with only 14 reports since 1959. The authors report the existence of a family lacking any stigmata of NF2, in which two members had spinal meningiomas. Tumor specimens were subjected to immunocytochemical analysis for the NF2 protein product Merlin, which has been implicated in the tumorigenesis of meningioma. Merlin immunoreactivity was present in both tumor specimens, implying that the NF2 tumor suppressor gene was not deleted in these tumors. This supports the hypothesis that a second tumor suppressor gene locus, other than NF2, acts in the formation of familial sporadic meningioma. The results are discussed in the context of putative oncogenic mechanisms of familial meningiomas.

Systemic delivery of human growth hormone or human factor IX in dogs by reintroduced genetically modified autologous bone marrow stromal cells.

Canine bone marrow stromal cells were expanded to numbers in excess of 10(9) cells from the initial 10-20 ml of marrow aspirates and transfected to express high levels of human growth hormone (hGH) in vitro. Ex vivo-modified marrow stromal cells were used in a gene therapy model system for the systemic delivery of transgene products in dogs. Adherent bone marrow stromal cell cultures, established and expanded from iliac crest marrow aspirates from each of 8 dogs, were transfected with a hGH gene plasmid expression vector and shown to express from 0.54-3.84 micrograms/10(6) cells per 24 hr hGH in vitro. The transfected plasmid vector does not possess a eukaryotic origin of replication nor does it possess sequences required for efficient integration into the host cell genome. As such, expression was expected to be transient. Transfected cells were autologously reintroduced into each dog by either infusion into a foreleg vein or directly into iliac crest marrow. In two cases, the stromal cells were cryopreserved following transfection, and subsequently thawed and infused. In one case, the expanded stromal cells were first cryopreserved, and then thawed, recultured, transfected, and infused. Reintroduced cell numbers ranged from 2.2 x 10(7) to 2.6 x 10(9), with total hGH expression capacities ranging from 62 to 1,400 micrograms/24 hr. Plasma of each of the dogs contained detectable hGH for a mean of 3.1 days (SD +/- 0.8 day) ranging from 2 to 5 days following reinfusion of cells. Peak plasma levels ranged from 0.10 to 1.76 ng/ml. Similar hGH expression values, based upon total expression capacity of the cells infused and dog body weight, were obtained for all dogs. Vector-modified stromal cells were detectable, by polymerase chain reaction (PCR) analysis, in the peripheral circulation following reinfusion in all 4 dogs analyzed. In 3 of the dogs, modified stromal cells were detected for 8.5-15 weeks. In addition, modified stromal cells were detected in iliac crest marrow of 2 dogs for 9 and 13 weeks, respectively, following reinfusion. In another experiment, cultured bone marrow stromal cells were transfected with a human factor IX (hFIX) plasmid vector. Modified cells (5.57 x 10(8)), with a total hFIX expression capacity of 281 micrograms/24 hr, were reinfused, resulting in detectable hFIX in plasma continuously for 9 days with a peak level of 8 ng/ml on day 1. These results demonstrate that the ex vivo bone marrow stromal cell system is a potentially powerful method by which to deliver secreted transgene product to the systemic circulation of large animals.

Synthesis of inositol 2-phosphate-quercetin conjugates.

The antiproliferative flavonoid, quercetin, is limited in its pharmacological utility by its low water solubility. In this paper, we describe the synthesis of two quercetin analogues prepared by linking the hydroxyl group at the 3- or 5-position of the flavonoid to the 1-hydroxyl group of myo-inositol-2-phosphate via a succinate diester linkage. The resulting conjugates were found to have dramatically enhanced water solubility relative to quercetin; the 5-linked quercetin analogue 2 had a water solubility of > 300 mg/mL at 20 degrees C. Comparison of the in vitro cytotoxicity and antiproliferative activity of conjugate 2 with those of quercetin toward cultured human colon adenocarcinoma (SW480) and human glioblastoma (U87MG) cells indicated that this modification of quercetin does not significantly diminish its activity in these assays.

Regulated expression of APE apurinic endonuclease mRNA during wound healing in porcine epidermis.

Abasic (AP) sites in DNA are cytotoxic and mutagenic and their repair is initiated by AP endonucleases. The major AP endonuclease of mammalian cells is encoded by the APE gene. Ape protein has also been proposed to modulate the activity of some transcription factors independently of its AP endonuclease activity. We investigated whether APE expression is coordinated with cell division, which could diminish mutagenesis. The level of APE mRNA was followed during wound healing in porcine epidermis, in which surgical wounding prompts rapid cell proliferation followed by a differentiation program to regenerate normal skin. In situ hybridization with a probe from human APE cDNA revealed strongly decreased expression in rapidly proliferating migrating cells during the first 1-3 days following wounding, succeeded by sharply increased APE expression that exceeded the pre-wounding levels by days 9-17. These changes were not observed in the surrounding undamaged tissue. In contrast to the foregoing in vivo results, APE expression in cultured primary human fibroblasts (IMR90) or myeloid leukemia cells (K562) was not coordinated with cell division. This biphasic APE expression during wound healing could relate to transcription factor regulation or it could allow unhindered DNA synthesis or prepare the developing epidermis to handle DNA damage. However, if transient under-expression of APE-encoded repair enzyme does occur, it might render regenerating skin especially vulnerable to mutagenesis during the cell proliferation phase.

Cell-cycle regulator cyclin D1 mRNA and protein overexpression occurs in primary malignant gliomas.

Gliomas are malignant brain tumors thought to arise through multi-step tumorigenesis, involving both the activation of oncogenes and the loss of tumor suppressor genes. The cyclin D1 gene encodes a proto-oncogenic cell cycle regulator implicated in the pathogenesis of several types of cancer, including gliomas. Northern blot analysis revealed expression of cyclin D1 mRNA in 7 (47%) of 15 primary glioma specimens. Immunohistochemistry using an antibody specific for cyclin D1 showed strong positivity amongst neoplastic glial cells in the same glioma samples. No cyclin D1 mRNA or protein was detected in 5 normal brain specimens. Cyclin B, which has not been linked to tumorigenesis and serves as a marker for cellular proliferation, was expressed in all tumor, but not control, samples. These data provide the first evidence for the overexpression of cyclin D1 mRNA and protein in primary human gliomas, and are consistent with a proposed oncogenic role of cyclin D1 in these tumors. It is suggested that excessive levels of cyclin D1 with or without several other D-type cyclin proteins may lead to deregulation of G(1) control in subsets of human gliomas. These results are further discussed in the context of putative functional redundancy of D-type cyclins and the role of the D-type cyclin/p16-ink4/pRB pathway in tumorigenesis.

Coexpression of EGF receptor and TGF alpha mRNA and protein occurs in primary meningiomas.

Meningiomas are benign brain tumors thought to arise by multi-step tumorigenesis, involving both the activation of proto-oncogenes and the loss of tumor suppressor genes. The EGFR gene encodes a protooncogenic tyrosine kinase growth factor receptor implicated in the pathogenesis of several types of cancer, including gliomas. TGF alpha, the ligand for EGFR, is thought to act through autocrine stimulation of EGFR present on the same and adjacent cells. Northern blot analysis revealed expression of EGFR mRNA in 9 of 11 (82%), and TGF alpha mRNA in 2 of 11 (18%), primary meningioma specimens. III situ hybridization verified EGFR and TGF alpha gene expression by meningioma cells. Immunocytochemistry using specific antibodies for EGFR and TGF alpha showed strong positivity amongst meningeal cells in the same meningioma samples. No EGFR or TGF alpha protein was detected in a sample of normal meninges. These data indicate that the autocrine coexpression of EGFR and TGF alpha mRNA and protein may occur in only a small proportion of primary meningiomas.

Expression of cyclin D1 proto-oncogene mRNA in primary meningiomas may contribute to tumorigenesis.

Meningiomas are benign brain tumors thought to arise by multi-step tumorigenesis, involving both the activation of oncogenes and the loss of tumor suppressor genes. The cell cycle regulator proto-oncogene cyclin D1 has been implicated in the pathogenesis of several types of cancer. Northern blot analysis revealed expression of cyclin D1 mRNA in 8 (53%), and cyclin B mRNA in 12 of 14 (86%), primary meningiomas. Immunocytochemistry using an antibody specific for cyclin D1 showed strong positivity amongst meningeal cells in the same meningioma samples. No cyclin D1 mRNA was detected in a sample of normal pachymeninges. Cyclin B, which has not yet been linked to tumorigenesis and serves as a marker for cellular proliferation, was expressed in a higher proportion of meningioma samples. These data provide the first evidence for the overexpression of cyclin D1 and B mRNA and protein in primary human meningiomas, and are consistent with a proposed oncogenic role of cyclin D1 in tumorigenesis. Excessive levels of the cyclin D1 proto-oncogene may lead to deregulation of G1 control in a proportion of arachnoid cap cells leading to tumorigenesis.

Overexpression of the ros1 gene in primary human gliomas may contribute to malignant progression.

Gliomas are malignant brain tumors thought to arise through multi-step tumorigenesis, involving both the activation of oncogenes and the loss of tumor suppressor genes. The ros1 gene encodes a proto-oncogenic protein which has been implicated, by in vitro studies, in the pathogenesis of several types of cancer, including gliomas. Northern blot analysis revealed expression of ros1 mRNA in 3 (30%) of 10 primary glioma specimens. In situ hybridization localized ros1 mRNA transcripts to GFAP positive tumor cells and pericytes around capillaries. Immunohistochemistry using an antibody specific for ros1 demonstrated strong positivity amongst neoplastic glial cells in the same glioma samples. No ros1 mRNA or protein was detected in 5 normal brain specimens. These data provide the first evidence for the overexpression of ros1 mRNA and protein in primary human gliomas, and are consistent with a proposed oncogenic role of ros1 in these tumors.

Expression of the human apurinic endonuclease gene (ape) in normal and malignant-tissues.

Apurinic/apyrimidinic endonucleases initiate repair at sites of base loss produced by many carcinogens and maintain genetic stability by repairing abasic sites produced spontaneously. We examined whether expression of the main apurinic endonuclease of human cells, encoded by the APE gene, might be decreased in malignant cells and thus potentiate an elevated mutation rate. Northern blotting and in situ hybridisation were used to quantitate the level of APE mRNA in various normal tissues and in 24 different brain tumors. There were no differences in APE expression among the normal tissues, or between malignant astrocytomas and meningiomas and the surrounding normal brain tissues. This suggests that diminished expression of the apurinic endonuclease does not underly the induction of these cancers, or explain clinical variations in presentation and response to treatment.

p53 expression during normal tissue regeneration in response to acute cutaneous injury in swine.

The present studies investigated the in vivo expression of the p53 suppressor gene and protein product in response to acute cutaneous injury in swine, along with the parallel expression of the c-sis/PDGF-B mitogen and its receptor beta (PDGF-R beta). p53 expression was shown to be suppressed during the period of active cellular proliferation in the injured tissue and to reemerge during the stages of healing. In contrast, c-sis/PDGF-B and PDGF-R beta were expressed during the early phase of active cellular proliferation and they were suppressed upon healing. This inverse relationship between mitogenic growth factors and p53 suggests the presence of well-controlled physiologic mechanisms that regulate in vivo the processes of normal tissue repair in response to injury. At the stages of tissue regeneration, these mechanisms include both the expression of growth factors that promote cell proliferation and the suppression of p53 that downregulates proliferation. At the stages of healing, the expression of the mitogenic growth factors is suppressed and that of p53 reemerges, reaching its peak at the time of complete epithelialization and healing of the injured tissue. These studies are the first to link the response of p53 protein to physiologic processes of tissue regeneration in vivo.

Expression of growth factor and receptor mRNAs in skin epithelial cells following acute cutaneous injury.

We report that acute injury induces the expression of selective growth factor and growth factor receptors in the epithelial cells of the wounded tissue. In situ hybridization analysis of skin biopsy specimens obtained after cutaneous injury in swine demonstrated the induction of the expression of transforming growth factor-alpha, its receptor, epidermal growth factor-R, acidic fibroblast growth factor, and basic fibroblast growth factor messenger RNAs in the skin epithelial cells of the wounded tissue. There was no significant expression in the epithelial cells of control, uninjured tissues. The expression levels were maximal during the period of active tissue repair (1 to 5 days after injury) and were totally suppressed upon the healing of the wounded tissues. In contrast, insulinlike growth factor-I, (IGF-I), IGF-I receptor, and IGF-II receptor messenger RNAs were expressed in the epithelial cells of both the control, uninjured tissues and in tissue specimens obtained after injury. There was no significant expression of IGF-II messenger RNA in the epithelial cells before or after injury. It seems that injury induces the coordinated expression of selective growth factor and growth factor receptor genes whose products contribute to the regulation of the complex processes involved in tissue repair and remodeling.

Expression of androgen and progesterone receptors in primary human meningiomas.

Meningiomas are common brain tumors that show a predilection for females and become more aggressive during pregnancy and menses. The existence of gender-specific hormone receptors in meningiomas has long been a matter of controversy; the recent cloning of androgen, estrogen, and progesterone receptors has facilitated their direct evaluation. The authors have demonstrated the expression of androgen and progesterone receptor messenger ribonucleic acid and protein product in nine primary human meningiomas by Northern blot analysis. Cellular localization was achieved by in situ hybridization analysis. Estrogen receptor expression was not detected. Normal adult meninges were shown to express very low levels of both androgen and progesterone receptors.

Human prostate adenocarcinomas express in-vivo messenger-rnas and protein products for platelet-derived growth factor-B and its receptor.

In situ hybridization and immunocytochemistry studies have shown the in vivo expression of platelet-derived growth factor B (PDGF-B) and PDGF receptor (PDGF-R) beta mRNAs and their respective protein products in the malignant epithelial cells of eight primary human prostatic adenocarcinomas. Examination of five nonmalignant adjacent prostate tissues did not demonstrate significant expression of PDGF B and PDGF-R beta mRNAs or production of their respective protein products in nonmalignant epithelial cells. Expression of androgen receptor mRNA was shown to be present in the epithelial cells of all of the five nonmalignant adjacent prostate tissues. There was a significant reduction in the expression of androgen receptor mRNA in poorly differentiated regions, and a moderate reduction in the well differentiated regions of the malignant tissues. It appears that dedifferentiation of the tumor cells in prostatic adenocarcinomas is accompanied by a reduction in androgen receptor mRNA expression. The coexpression of PDGF and its receptor in the malignant epithelial cells of prostatic adenocarcinomas signifies an abnormal autocrine loop that may contribute to their growth and maintenance.

Expression of monocyte chemoattractant protein 1 mRNA in human idiopathic pulmonary fibrosis.

Macrophages are thought to play an important role in the pathologic changes associated with idiopathic pulmonary fibrosis (IPF). The mechanisms for increased monocyte/macrophage recruitment in IPF are unknown. Monocyte chemoattractant protein 1 (MCP-1) is the predominant monocyte chemoattractant secreted by a variety of different cell types in culture. We examined the expression of MCP-1 mRNA and its protein product in vivo in IPF and non-IPF lung specimens by in situ hybridization and immunocytochemistry. The cell types expressing MCP-1 in vivo were identified by immunostaining with specific antibodies. We demonstrated the expression of MCP-1 mRNA in pulmonary epithelial cells, in monocytes/macrophages, and in vascular endothelial and smooth muscle cells. Lung epithelial cells in patients with IPF strongly expressed MCP-1 mRNA and its protein product. In contrast, epithelial cells in non-IPF specimens did not express MCP-1 mRNA. Macrophages and vascular endothelial and smooth muscle cells were shown to express MCP-1 in both IPF and non-IPF lung specimens. These findings provide a basis for the understanding of the in vivo physiologic processes that mediate monocyte/macrophage recruitment and infiltration in the lung interstitium and the pathologic state contributing to an increased alveolar monocyte/macrophage population and inflammation in IPF.

Effect of the expression of transforming growth factor-beta 2 in primary human glioblastomas on immunosuppression and loss of immune surveillance.

Glioblastomas are malignant brain tumors that are attended by an immunosuppressed state. The authors have studied the expression of transforming growth factor-beta 2, which is known to have potent immunosuppressive and angiogenic properties. Transforming growth factor-beta 2 messenger ribonucleic acid and its protein product are both found to be greatly overexpressed in these tumors and are absent from normal brain tissue. The overexpression of this growth factor may contribute to the escape of neoplastic astrocytes from immune surveillance and, furthermore, to the immunosuppressed state that is characteristic of many of these patients.

Malignant epithelial cells in primary human lung carcinomas coexpress in vivo platelet-derived growth factor (PDGF) and PDGF receptor mRNAs and their protein products.

Lung cancer represents one of the major human carcinomas with the highest degree of mortality. Epidemiologic studies have linked this disease to "chronic injury," largely induced by cigarette smoking. In the present studies, we demonstrate the in vivo expression of platelet-derived growth factor (PDGF) and PDGF receptor (PDGF-R) beta mRNAs and their respective protein products in malignant epithelial cells of primary human lung carcinomas. In contrast, nonmalignant epithelial cells in control, normal lung tissue specimen did not express PDGF and PDGF-R mRNAs and did not produce their respective protein products. Epithelial cells in lung specimen from patients with idiopathic pulmonary fibrosis expressed only PDGF mRNA but not PDGF-R beta mRNA. These findings of the inappropriate coexpression of a potent mitogen, PDGF, and its receptor in lung cancer epithelial cells suggest the presence of a powerful in vivo mechanism contributing to the self-stimulation and unregulated growth of lung cancer tumor cells.

Expression of insulin-like growth factors I and II and their receptor mRNAs in primary human astrocytomas and meningiomas; in vivo studies using in situ hybridization and immunocytochemistry.

These studies demonstrate the expression of IGF-1, IGF-11, and their respective receptor mRNAs in primary human astrocytomas and meningiomas. In situ hybridization and immunocytochemistry have localized a strong expression of both IGF-1 and IGF-11 mRNAs and of their protein products in the tumor cells of astrocytomas and meningiomas. The expression of IGF-1 and IGF-11 mRNAs in the tumor cells was accompanied by the co-expression of their respective type-1 and type-11 IGF receptor mRNAs. Control, non-malignant human brain expressed IGF-1 mRNA and IGF-1 and IGF-11 receptor mRNAs. There was no significant expression of IGF-11 MRNA in the control brain specimens. Control pachymeninges (dura mater) expressed low levels of IGF-1 mRNA and IGF-1 receptor mRNA. There was no significant expression of IGF-11 and IGF-11 receptor mRNAs in pachymeninges. The co-expression of IGFs and their receptors in brain tumors may contribute in their development and maintenance. The strong inappropriate expression of IGF-11 mRNA and its protein product in the tumor cells of astrocytomas and meningiomas, but not in normal brain specimens, may serve as molecular markers for the early detection of these tumors.

Expression of monocyte chemotactic protein-1 in human melanoma in vivo.

A common feature of human melanoma is infiltration by monocytes at early stages of tumorigenesis. This infiltration may be highly significant since macrophages have the capacity to alter the behavior of tumor cells. The authors previously demonstrated that the predominant monocyte chemoattractant produced by tumor cells in vitro was monocyte chemotactic protein-1 (MCP-1). The authors identify the expression of MCP-1 in pathologic specimens of both primary and metastatic human melanoma but not in normal skin. The finding that MCP-1 is produced by malignant melanoma suggests that specific genes are expressed in tumor cells that can induce the recruitment of monocytes in vivo.