FGF-18, a novel member of the fibroblast growth factor family, stimulates hepatic and intestinal proliferation.

The fibroblast growth factors (FGFs) play key roles in controlling tissue growth, morphogenesis, and repair in animals. We have cloned a novel member of the FGF family, designated FGF-18, that is expressed primarily in the lungs and kidneys and at lower levels in the heart, testes, spleen, skeletal muscle, and brain. Sequence comparison indicates that FGF-18 is highly conserved between humans and mice and is most homologous to FGF-8 among the FGF family members. FGF-18 has a typical signal sequence and was glycosylated and secreted when it was transfected into 293-EBNA cells. Recombinant murine FGF-18 protein (rMuFGF-18) stimulated proliferation in the fibroblast cell line NIH 3T3 in vitro in a heparan sulfate-dependent manner. To examine its biological activity in vivo, rMuFGF-18 was injected into normal mice and ectopically overexpressed in transgenic mice by using a liver-specific promoter. Injection of rMuFGF-18 induced proliferation in a wide variety of tissues, including tissues of both epithelial and mesenchymal origin. The two tissues which appeared to be the primary targets of FGF-18 were the liver and small intestine, both of which exhibited histologic evidence of proliferation and showed significant gains in organ weight following 7 (sometimes 3) days of FGF-18 treatment. Transgenic mice that overexpressed FGF-18 in the liver also exhibited an increase in liver weight and hepatocellular proliferation. These results suggest that FGF-18 is a pleiotropic growth factor that stimulates proliferation in a number of tissues, most notably the liver and small intestine.

JNK1, JNK2 and JNK3 are p53 N-terminal serine 34 kinases.

The function of the tumor suppressor protein p53 is modulated by post-translational events, primarily by phosphorylation. p53 is phosphorylated at multiple sites by a variety of protein kinases depending on the cellular environment. It has been suggested that serine 34 of mouse p53 is specifically phosphorylated by a stress-activated protein kinase in response to ultraviolet radiation. Since serine 34 is a major site of phosphorylation of mouse p53 in vivo and its specific protein kinase is still not definitively identified yet, we have examined the c-Jun N-terminal kinase 1 (JNK1) activity on p53 by expressing JNK1 in 293T cells. We show here that activated JNK1 phosphorylates mouse p53 specifically at serine 34 in vitro, while a dominanant-negative JNK1 mutant does not phosphorylate p53. More importantly, JNK1 associates with p53 in vivo, with or without activation, confirming that JNK1 is indeed a p53 kinase. Interestingly, activated JNK2 and JNK3 also phosphorylate serine 34 of mouse p53. Furthermore, JNK2 and JNK3 also associate with p53 in vivo, indicating that not only JNK1, but also JNK2 and JNK3 are p53 N-terminal serine 34 kinases. Phosphorylation of p53 by JNKs may play an important role in nuclear signal transduction in response to environmental stress or tumorigenic agents.

Human fibroblast growth factor-18 stimulates fibroblast cell proliferation and is mapped to chromosome 14p11.

The fibroblast growth factors (FGFs) play crucial roles in controlling embryonic development, cell growth, morphogenesis, and tissue repair in animals. Furthermore, FGFs may have a role in angiogenesis and may be involved in tumor invasion and metastasis. Here, we present the cloning and sequence of human FGF-18, a novel member of the FGF family. Sequence comparison indicates that FGF-18 is conserved with the other FGFs and most homologous to FGF-8 among the FGF family members. We showed that human FGF-18 was expressed primarily in the heart, skeletal muscle, and pancreas, and at lower levels in the other tissues. FGF-18 was also expressed at low levels in certain cancer cell lines. FGF-18 contains a typical signal peptide and was secreted when it was transfected into mammalian cells. Recombinant FGF-18 protein stimulated proliferation in the fibroblast cell line NIH3T3 in a dose-dependent manner, suggesting that FGF-18 is a functional growth factor. Finally, the FGF-18 gene was evolutionarily conserved, and localized to human chromosome 14p11.

Hematopoietic progenitor kinase-1 (HPK1) stress response signaling pathway activates IkappaB kinases (IKK-alpha/beta) and IKK-beta is a developmentally regulated protein kinase.

Nuclear factor kappa-B (NF-kappaB) is a pleiotropic transcription factor that plays a central role in the immune and inflammatory responses, and is also involved in controlling viral transcription and apoptosis. A critical control in the activation of NF-kappaB is the phosphorylation of its inhibitory factor IkappaBs by IkappaB kinases (IKK-alpha and -beta). Here, we present experiments addressing the regulation and global expression of murine IKK-beta, and localize the IKK-beta gene to mouse chromosome 8A3-A4. IKK-beta was expressed primarily in the liver, kidney and spleen, and at lower levels in the other adult tissues. While IKK-beta was expressed ubiquitously throughout the mouse embryo at 9.5 days, its expression began to be localized to the brain, neural ganglia, neural tube, and liver in the 12.5-day's embryo. At 15.5 days, the expression of IKK-beta was further restricted to specific tissues of the embryo, suggesting that IKK-beta is a developmentally regulated protein kinase. Interestingly, IKK-beta phosphorylated IkappaB constitutively, whereas IKK-alpha was not active in the absence of cell stimulation. Moreover, both IKK-alpha and -beta were activated by hematopoietic progenitor kinase-1 (HPK1) and MAPK/ERK kinase kinase-1 (MEKK1) specifically, suggesting that IkappaB/NF-kappaB is regulated through the HPK1-MEKK1 stress response signaling pathway.

The Rb gene suppresses the growth of normal cells.

The suppression of tumor formation, first demonstrated by somatic cell hybrid and microcell fusion experiments, suggests the existence of a class of genes that selectively suppress the growth of tumor cells but not normal cells. The reintroduction of these genes into tumor cells presumably renders the cells responsive to in vivo growth inhibitory environment. As the inheritance of a defective retinoblastoma gene (Rb-1) allele results in a predisposition to the development of various cancers, and since inactivation of both alleles are observed in tumor cells, the Rb gene has been suspected to have the ability to suppress tumor growth. Data presented here demonstrated that different types of normal cells, which have a limited life span, were also growth arrested by a transfected Rb gene. Cell lines which are resistant to the growth suppression effect of the Rb gene in vitro, retain the ability to form tumors in nude mice even in the presence of a stable and highly expressed wild type Rb protein. We conclude that while the Rb gene can suppress the growth of many tumor cell lines, its growth suppression effect is not tumor specific.

[Kinetic observation and analysis of the radioactivity distribution of 125I-labelled F(ab')2 fragments of monoclonal antibody against human lung cancer cells in normal mice].

Radioactivity distribution of 125I-labelled F(ab')2 fragments in 14 organs and tissues of normal mice for 11 time-phases from 0.5 hour to 7 days after injection was observed. The radioactivity-time curves of these organs and tissues were divided into three types according to their characteristics: excreting type (blood and liver), absorbing type (thyroid), absorbing-excreting type (the other organs and tissues). The organs fully perfused with blood had shorter peak-time and higher peak-value, such as heart, lung, kidney and liver. All of the peak-times were equal to or less than 2 days after injection, therefore the image taking should be kept away from this period. The excreting rate constants of slow metabolic component were similar for various organs and tissues except thyroid and brain. The brain had a low peak in the curve and small excreting rate constant. It was demonstrated that F(ab')2 fragments were able to penetrate the blood-brain barrier but absorption and excretion were slower compared with the other organs and tissues. The radioiodine was obviously concentrated in the subcutaneous tissue, which may have been the main cause of the subcutaneous tissue tumor induced by radioiodine. The radioactivity-time curve in which the peak value and inflect point existed simultaneously shows that radioactivity metabolism after injection of 125I-F(ab')2 follows three compartment models. The biological meanings corresponding to three sections of the blood radioactivity-time curve are pure distribution, distribution-recycle and catabolic-excretory phases, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

[Reactivity of monoclonal antibodies ALT-01 and ALT-04 and identification of lung cancer-associated antigens].

Two IgG1 type monoclonal antibodies ALT-01 and ALT-04 were prepared by two different immunization schedules. ALT-01 was generated by fusing murine myeloma NS-1 cells with splenocytes from a BALB/c mouse immunized by human lung squamous carcinoma cells, which were coated by antisera to mixed human lymphocytes. For preparation of ALT-04, human lung squamous carcinoma xenograft-bearing nude mice were injected I. P. with the spleen cells of normal BALB/c mice in order to acquire immunofunction. The spleen cells from these tumor-bearing nude mice were fused with NS-1 cells. Then, these hybridomas were screened and cloned for 3 times. Two antibodies were shown to recognize the surface antigen on human lung carcinoma cells and several kinds of tumor cell lines but not those on normal cell lines. ALT-01 reacted to neither human lung carcinoma tissue nor its xenograft. ALT-04 reacted to human lung carcinoma tissue, of which, reaction to adenocarcinoma was the strongest but not to various normal tissues. Immunoprecipitation followed by SDS-polyacrylamide gel electrophoresis and autoradiography was used to detect the associated antigen in 35S-labeled human lung carcinoma cells. Antigens, reacting to ALT-01, show one band of Mr 38,000 but those to ALT-04 reveal two bands of Mr 48,000 and 36,000.

[Radioimmunodetection of human lung cancer xenografts by isotope labeling monoclonal antibody ALT-04 in nude mice].

Three nude mouse models bearing the human lung cancer were established from two fresh surgical specimens and one cell line. Tumor histological structure and cell morphology were similar before and after transplantation. The monoclonal antibody ALT-04 (McAb ALT-04) against human lung cancer was labeled by 125I, 131I and 201Tl. Radioimmunodetection study showed that all the three kinds of xenografts in nude mice were specifically located by McAb ALT-04. Imaging examination confirmed the ability of isotope labeling McAb ALT-04 to detect the presence of transplanted human lung cancer tissues without the aid of background subtraction manipulations. It is suggested that McAb ALT-04 have the possibility of locating in the tumor diagnosis and guiding in the treatment.

Human pregnancy-specific beta 1 glycoprotein is encoded by multiple genes localized on two chromosomes.

A human genomic library was screened with a mixture of two cDNA probes, with one covering the 5' coding sequence and the other containing the 3'-end portion of human pregnancy-specific beta 1 glycoprotein (SP1). Seventeen clones were identified, all of which carried insert fragments capable of hybridizing with the cDNA probe. Insert size of these clones varied from 15.0 to 19.8 kb. Partial restriction maps were constructed, which demonstrated the presence of at least seven groups of unique SP1 genomic clones and suggested the possibility of multiple genes coding for SP1. The multigene nature of SP1 was confirmed by hybridization of the SP1 cDNA probe to multiple bands on Southern blots of human genomic DNA. Further analysis with chromosomal DNA dot blot demonstrated the presence of homologous sequences on the X chromosome and autosomal chromosome 6. Thus, human SP1 is apparently coded for by more than one gene residing on the X and 6 chromosomes.

Human HPK1, a novel human hematopoietic progenitor kinase that activates the JNK/SAPK kinase cascade.

The c-Jun amino-terminal kinases (JNKs)/stress-activated protein kinases (SAPKs) play a crucial role in stress responses in mammalian cells. The mechanism underlying this pathway in the hematopoietic system is unclear, but it is a key in understanding the molecular basis of blood cell differentiation. We have cloned a novel protein kinase, termed hematopoietic progenitor kinase 1 (HPK1), that is expressed predominantly in hematopoietic cells, including early progenitor cells. HPK1 is related distantly to the p21(Cdc42/Rac1)-activated kinase (PAK) and yeast STE20 implicated in the mitogen-activated protein kinase (MAPK) cascade. Expression of HPK1 activates JNK1 specifically, and it elevates strongly AP-1-mediated transcriptional activity in vivo. HPK1 binds and phosphorylates MEKK1 directly, whereas JNK1 activation by HPK1 is inhibited by a dominant-negative MEKK1 or MKK4/SEK mutant. Interestingly, unlike PAK65, HPK1 does not contain the small GTPase Rac1/Cdc42-binding domain and does not bind to either Rac1 or Cdc42, suggesting that HPK1. activation is Rac1/Cdc42-independent. These results indicate that HPK1 is a novel functional activator of the JNK/SAPK signaling pathway.

Murine p38-delta mitogen-activated protein kinase, a developmentally regulated protein kinase that is activated by stress and proinflammatory cytokines.

The p38 mitogen-activated protein kinases (MAPK) play a crucial role in stress and inflammatory responses and are also involved in activation of the human immunodeficiency virus gene expression. We have isolated the murine cDNA clones encoding p38-delta MAPK, and we have localized the p38-delta gene to mouse chromosome 17A3-B and human chromosome 6p21.3. By using Northern and in situ hybridization, we have examined the expression of p38-delta in the mouse adult tissues and embryos. p38-delta was expressed primarily in the lung, testis, kidney, and gut epithelium in the adult tissues. Although p38-delta was expressed predominantly in the developing gut and the septum transversum in the mouse embryo at 9.5 days, its expression began to be expanded to many specific tissues in the 12.5-day embryo. At 15.5 days, p38-delta was expressed virtually in most developing epithelia in embryos, suggesting that p38-delta is a developmentally regulated MAPK. Interestingly, p38-delta and p38-alpha were similar serine/threonine kinases but differed in substrate specificity. Overall, p38-delta resembles p38-gamma, whereas p38-beta resembles p38-alpha. Moreover, p38-delta is activated by environmental stress, extracellular stimulants, and MAPK kinase-3, -4, -6, and -7, suggesting that p38-delta is a unique stress-responsive protein kinase.

Protein phosphatase X interacts with c-Rel and stimulates c-Rel/nuclear factor kappaB activity.

Nuclear factor kappaB (NF-kappaB) and the Rel family of proteins are pleiotropic transcription factors that play central roles in the immune and inflammatory responses, as well as apoptosis. Here, we identified a serine/threonine protein phosphatase X (PPX; also called protein phosphatase 4 (PP4)) that specifically associated with c-Rel, NF-kappaB p50, and RelA. The amino acid sequences of human and mouse PPX are 100% identical, and the PPX gene was mapped to human chromosome 16 p11.2. Overexpression of PPX, but not catalytically inactive PPX mutants, stimulated the DNA-binding activity of c-Rel and activated NF-kappaB-mediated transcription. These results suggest that PPX is a novel activator of c-Rel/NF-kappaB.

APRIL and TALL-I and receptors BCMA and TACI: system for regulating humoral immunity.

We report that the tumor neurosis factor homolog APRIL (a proliferation-inducing ligand) stimulates in vitro proliferation of primary B and T cells and increases spleen weight due to accumulation of B cells in vivo. APRIL functions via binding to BCMA (B cell maturation antigen) and TACI (transmembrane activator and CAML-interactor) and competes with TALL-I (also called BLyS or BAFF) for receptor binding. Soluble BCMA and TACI specifically prevent binding of APRIL and block APRIL-stimulated proliferation of primary B cells. BCMA-Fc also inhibits production of antibodies against keyhole limpet hemocyanin and Pneumovax in mice, indicating that APRIL and/or TALL-I signaling via BCMA and/or TACI are required for generation of humoral immunity. Thus, APRIL-TALL-I and BCMA-TACI form a two ligands-two receptors pathway involved in stimulation of B and T cell function.