Neuroendocrine lung tumors: grade correlates with proliferation but not angiogenesis.

Angiogenesis has been implicated in the progression of human neoplasia from benign precursor to invasive and metastatic phenotypes. The acquisition of dominant oncogenes in preneoplastic cells in vitro and in vivo has been associated with the increased ability of tumor cells to secrete angiogenic mediators and recruit blood vessels. However, in a subset of benign lesions, high levels of angiogenesis have been found before the conversion to invasive and metastatic phenotypes. In many of these benign lesions, dominant oncogenic pathways are activated first; then as malignant potential is acquired, there is a loss of nuclear tumor suppressor genes, such as p53 and p16. We studied neuroendocrine lung tumors (NLT) ranging from typical and atypical carcinoid tumors to large cell neuroendocrine and small cell carcinomas in order to determine whether angiogenesis (as assessed by mean vessel density) and proliferation rates (as assessed by MIB-1 nuclear immunohistochemical staining) correlate with tumor type. We found that increased rates of proliferation, but not angiogenesis, correlate with tumor type. The association of increased proliferation and tumor type may prove to be clinically useful and shed light on the role of sequential oncogenic alterations in NLT.

Consultative (expert) second opinions in soft tissue pathology. Analysis of problem-prone diagnostic situations.

We reviewed 500 consecutive soft tissue lesions referred for expert consultation to determine types of lesions and/or situations in which major discrepancies occur. Of 266 cases (53.2%) accompanied by a diagnosis, essential agreement with the second opinion was noted in 68%, minor discrepancy in 7%, and major discrepancy in 25%. The 65 major discrepancies were distributed proportionally to the referring sources and could be divided into 4 groups: benign mesenchymal lesions diagnosed as sarcomas (45%), sarcomas diagnosed as benign tumors (23%), nonmesenchymal lesions diagnosed as sarcoma (20%), and major grading discrepancies (12%). Relatively few lesions accounted for a major proportion of major discrepancies. Problematic lesions were lipoma and fasciitis and their variants and desmoplastic-neurotropic melanoma. Needle biopsy specimens were somewhat more likely to be associated with a discrepant opinion. With the exception of nonmesenchymal lesions, the diagnosis for all major discrepant cases could be made on the basis of the H&E-stained slides, suggesting that failure to perform immunostains did not account for discrepancies. Lack of familiarity with rare or unusual lesions is probably more significant in explaining diagnostic discrepancies than is the increasing use of needle biopsy or the failure to perform immunohistochemical analysis.

The generation and characterization of a cell line derived from a sporadic renal angiomyolipoma: use of telomerase to obtain stable populations of cells from benign neoplasms.

Angiomyolipomas are benign tumors of the kidney derived from putative perivascular epithelioid cells, that may undergo differentiation into cells with features of melanocytes, smooth muscle, and fat. To gain further insight into angiomyolipomas, we have generated the first human angiomyolipoma cell line by sequential introduction of SV40 large T antigen and human telomerase into human angiomyolipoma cells. These cells show phenotypic characteristics of angiomyolipomas, namely differentiation markers of smooth muscle (smooth muscle actin), adipose tissue (peroxisome proliferator-activator receptor gamma, PPARgamma), and melanocytes (microophthalmia, MITF), thus demonstrating that a single cell type can exhibit all of these phenotypes. These cells should serve as a valuable tool to elucidate signal transduction pathways underlying renal angiomyolipomas.

Differential expression of active mitogen-activated protein kinase in cutaneous endothelial neoplasms: implications for biologic behavior and response to therapy.

BACKGROUND: Tumors of endothelium range from benign hemangiomas of infancy to highly malignant angiosarcomas of the elderly. Hemangiomas are the most common tumors in infants and may affect up to 10% of all children. The biologic behavior of these lesions ranges from self-resolving, in the case of hemangiomas and pyogenic granulomas, to lethal metastatic neoplasms in the case of angiosarcoma. Although the clinical outcomes of these diseases are easily distinguished, the biologic basis for these differences is not well understood. Activation of mitogen-activated protein kinase (MAPK) is an important signal transduction mechanism that may predict response of a tumor to chemotherapy. OBJECTIVE: Our purpose was to examine expression of phosphorylated (activated) MAPK in hemangiomas of infancy, pyogenic granulomas, hemangioendotheliomas, and angiosarcomas to determine whether phosphorylated MAPK was expressed in endothelial tumors. In addition, we examined endothelial tumors of infectious origin, Kaposi's sarcoma, and verruga peruana. METHODS: Skin sections from benign and malignant endothelial tumors, including hemangioma of infancy, angiosarcoma, and infectious endothelial lesions (Kaposi's sarcoma, verruga peruana) were stained with an antibody specific for phosphorylated MAPK. RESULTS: We demonstrated strong expression of phosphorylated MAPK in benign endothelial tumors, including capillary hemangioma of infancy and pyogenic granuloma, and greatly decreased expression in angiosarcoma. In addition, infectious endothelial tumors stained strongly with this antibody, similar to benign tumors. The presence of immunoreactive phosphorylated MAPK appears to be inversely correlated with degree of malignancy. CONCLUSION: We demonstrate that the use of antibodies specific for signal transduction pathways is feasible in paraffin-fixed tissue. Thus the activity of a given signal transduction pathway can be ascertained in a biopsy specimen. Immunohistochemistry for phosphorylated MAPK may help the pathologist distinguish benign from malignant endothelial processes and thus guide therapy.

Vasopressin messenger ribonucleic acid regulation via the protein kinase A pathway.

Activation of vasopressin (VP) gene expression in vivo by osmotic stimuli results in an increase in both messenger RNA (mRNA) content and polyadenylate [poly(A)] tail length. VP gene transcription in vitro is stimulated by protein kinase A (PKA) activation. To examine the role of PKA in the regulation of VP mRNA poly(A) metabolism, constructs of the rat VP gene were permanently transfected into the mouse anterior pituitary cell line, AtT-20. Treatment with forskolin of cells expressing the intact VP gene resulted in increased VP gene transcription, an increase in the content of VP mRNA, and a shift toward VP mRNA species with longer poly(A) tails accompanied by the loss of VP mRNA species with shorter poly(A) tails. We uncoupled the PKA-stimulated appearance of long-tailed species from the disappearance of short-tailed species, suggesting that the size shift was caused by a coincident, but uncoupled net increase in VP mRNA species with elongated poly(A) tails and net loss of mRNA species with short poly(A) tails. These data indicate that activation of the PKA second-messenger pathway both enhances transcription of the VP gene and causes an increase in the average length of VP mRNA poly(A) tails. This latter effect, by shifting upwards the average poly(A) tail size, could result in increased translational efficiency or stability of VP mRNA, thereby providing an additional mechanism by which PKA may enhance gene expression.

Regulation of the ectopically expressed human glycoprotein alpha-subunit gene in the human hepatoma cell line NPLC.

The human glycoprotein alpha-subunit is the common subunit of the heterodimeric hormones CG (hCG), TSH, LH, and FSH. Human glycoprotein alpha-subunit is produced eutopically in placenta, pituitary, and choriocarcinoma and ectopically in a large variety of human tumors. We report ectopic glycoprotein alpha-subunit messenger RNA (mRNA) and peptide production in the human hepatoma cell line, NPLC. Neither hCG beta mRNA nor intact hCG peptide was detected. Antimetabolite regulation of glycoprotein alpha-subunit expression in NPLC cells resembled that found in choriocarcinoma cells in that it was stimulated by hydroxyurea. In addition, glycoprotein alpha-subunit mRNA expression and transcription in NPLC were stimulated by activators of the protein kinase A and C second messenger pathways, as well as by glucocorticoid. Glucocorticoid augmented glycoprotein alpha-subunit gene transcription by phorbol ester and forskolin, in contrast to its simultaneous inhibitory effect on phorbol ester activation of the CRH gene, which is also ectopically expressed in these cells. Glucocorticoid thus modulates the activation of these genes by phorbol ester in opposite directions, despite their identical cellular context. The NPLC cell line provides a new model for the study of human glycoprotein alpha-subunit gene regulation and free glycoprotein alpha-subunit secretion. In addition, it should be useful for investigating the role that specific cis-acting DNA sequences play in glucocorticoid modulation of gene induction by second messenger pathways.

Effects of hydroxyurea and cyclic adenosine monophosphate/protein kinase A inhibitors on the expression of the human chorionic gonadotropin alpha subunit and c-myc genes in choriocarcinoma.

We have previously shown that treatment of choriocarcinoma cells with methotrexate or hydroxyurea leads to both cessation of cell growth, accompanied by repression of c-myc oncogene expression, and induction of genes associated with the placental phenotype, including both subunits of human chorionic gonadotropin (hCG) and placental alkaline phosphatase. Since the genes induced by these antimetabolites are also cyclic AMP inducible, we hypothesized that these antimetabolites may cause activation of the cyclic AMP/protein kinase A pathway, suppressing genes associated with cellular proliferation and inducing placental gene expression. Three inhibitors of the cyclic AMP/protein kinase A pathway were assayed for their ability to inhibit the induction of the human chorionic gonadotropin alpha gene by hydroxyurea, and none of these inhibitors eliminated this induction. In addition, blockade of the cyclic AMP/protein kinase A pathway did not reverse the suppression of c-myc by hydroxyurea. The results of the inhibitor studies suggest that hydroxyurea acts independently of the protein kinase A pathway to stimulate gene expression, and that suppression of c-myc is insufficient to cause the induction of the human chorionic gonadotropin alpha gene by hydroxyurea.

Differential regulation of choriocarcinoma gene expression by DNA synthesis inhibitors.

We have previously shown that methotrexate (MTX) and hydroxyurea (HU) stimulate expression of the human chorionic gonadotropin alpha and placental alkaline phosphatase genes and repress the expression of the c-myc oncogene in BeWo choriocarcinoma cells. In order to determine whether c-myc downregulation played a role in the induction of these placental genes, we treated BeWo choriocarcinoma cells with aphidicolin (APH), and 6-diazo-5-oxo-L-norleucine (DON), and compared the effects of these drugs with that of MTX. All of these drugs downregulate c-myc gene expression. At the doses used, both APH and DON repress c-myc expression to approximately the same extent, as well as inducing morphologic changes in BeWo similar to that of MTX, although neither stimulated hCG alpha expression. Both DON and APH stimulate placental alkaline phosphatase gene expression, but only MTX and DON stimulate cholesterol side chain cleavage enzyme gene expression. This indicates that the downregulation of c-myc gene expression is insufficient to stimulate the expression of all the placental genes stimulated by MTX.

SCPB-and FMRFamide-like immunoreactivities in lobster neurons: colocalization of distinct peptides or colabeling of the same peptide(s)?

Virtually all of the SCPB-like immunoreactive neurons (ca. 60 cells) in the lobster Homarus americanus also contain FMRFamide-like immunoreactivity. Control experiments reveal that SCPB-and FMRFamide-like immunoreactivities are successfully preadsorbed with their specific antigens, while the normal staining pattern is retained following preadsorption of each antibody with the alternate peptide. These experiments potentially lead to the conclusion that the anti-SCPB and anti-FMRFamide antibodies are labeling distinct compounds that are colocalized in lobster neurons. The lobster nervous system does not, however, contain authentic FMRFamide, but rather several FMRFamide-like compounds (Trimmer et al., J. Comp. Neurol. 266:16-26, 1987). The most abundant of these is the octapeptide TNRNFLRFamide. Experiments demonstrate that SCPB-like immunoreactivity is completely preadsorbed with synthetic TNRNFLRFamide, while there is a significant or complete loss of staining after preadsorption of the FMRFamide antibody with this molecule. Met-enkephalin-Arg-Phe-amide (YGGFMRFamide), an extended opioid peptide containing the FMRFamide sequence, also preadsorbs SCPB- and FMRFamide-like immunoreactivities, while Met-enkephalin-Arg-Phe (YGGFMRF) has no effect on the staining properties of these antibodies. These results suggest that the SCPB antibody can bind to extended forms of FMRFamide-like molecules, and that anti-SCPB and anti-FMRFamide antibodies may be colabeling one or more FMRFamide-like molecules in lobster neurons.

Regulation of gene expression in choriocarcinoma by methotrexate and hydroxyurea.

Choriocarcinoma, a highly malignant neoplasm of trophoblastic origin, is remarkable for its marked degree of sensitivity to chemotherapeutic agents. We treated two cell lines derived from choriocarcinoma patients with two antineoplastic agents, methotrexate and hydroxyurea (HU), both of which cause nucleotide depletion and have been previously shown to be effective against choriocarcinoma, and found pleiotropic regulation of several genes. Three genes, hCG alpha-subunit, beta-subunit, and placental alkaline phosphatase, were all strongly induced by methotrexate and HU. Expression of c-myc, an oncogene associated with proliferation, was reduced to nearly undetectable levels by the drug treatment, while expression of beta 2-microglobulin, a component of the class 1 major histocompatibility locus, was unchanged. In addition, the mechanism of induction by HU of one of these genes, the hCG alpha-subunit gene, was found to occur at the level of transcription. The similar effects of methotrexate and HU, two mechanistically unrelated antimetabolites, on the induction of specific gene expression in choriocarcinoma cells suggest that these effects are due to nucleotide pool alteration, rather than specific ligand effects. Furthermore, the hCG alpha-subunit promoter contains a transcriptionally regulated HU-responsive element.