The Fanconi anemia group C gene product modulates apoptotic responses to tumor necrosis factor-alpha and Fas ligand but does not suppress expression of receptors of the tumor necrosis factor receptor superfamily.

Exposure of hematopoietic progenitor cells (HPC) from mice and humans with Fanconi anemia group C (FAC) to interferon-gamma (IFN-gamma) or tumor necrosis factor-alpha (TNF-alpha) at doses too low to inhibit growth of normal HPC induces profound apoptotic responses. Because the IFN-gamma hypersensitivity of cells lacking the FAC protein is mediated, in part, through priming of the Fas pathway, and because several other members of this family are capable of inducing apoptosis either alone or in concert with each other, we tested the hypothesis that IFN-gamma induces increased expression of members of the TNF receptor (TNFR) superfamily in cells nullizygous for the FAC gene. Using isogenic human Epstein-Barr virus-transformed lymphoblast cell lines and c-kit+ bone marrow cells from mice with inactivating mutations of the FAC locus, we quantified mRNA levels by reverse transcriptase polymerase chain reaction and surface expression of the gene products by flow cytometry of TNFR1, TNFR2, Fas, CD30, CD40, and nerve growth factor receptor. We found that neither constitutive nor IFN-gamma-induced expression of these receptors was influenced by the absence of a functional FAC gene product, and expression of these receptors was not suppressed in nullizygous cells complemented with the normal FAC cDNA. We conclude that, although exaggerated apoptotic responses in FAC-deficient cells are at least partially mediated through activation of members of the TNFR superfamily, the normal FAC protein does not function as a direct suppressor of this family of molecules and inactivation of FAC does not augment expression of these proteins.

The Fanconi anemia group C gene product is located in both the nucleus and cytoplasm of human cells.

The Fanconi anemia (FA) complementation group C (FAC) protein gene encodes a cytoplasmic protein with a predicted Mr of 63,000. The protein's function is unknown, but it has been hypothesized that it either mediates resistance to DNA cross-linking agents or facilitates repair after exposure to such factors. The protein also plays a permissive role in the growth of colony-forming unit-granulocyte/macrophage (CFU-GM), burst-forming unit-erythroid (BFU-E), and CFU-erythroid (CFU-E). Attributing a specific function to this protein requires an understanding of its intracellular location. Recognizing that prior study has established the functional importance of its cytoplasmic location, we tested the hypothesis that FAC protein can also be found in the nucleus. Purified recombinant Escherichia coli-derived FAC antigens were used to create antisera able to specifically identify an Mr = 58,000 protein in lysates from human Epstein-Barr virus (EBV)-transformed cell lines by immunoblot analysis. Subcellular fractionation of the cell lysates followed by immunoblot analysis revealed that the majority of the FAC protein was cytoplasmic, as reported previously; however, approximately 10% of FAC protein was reproducibly detected in nuclear fractions. These results were reproducible by two different fractionation methods, and included markers to control for contamination of nuclear fractions by cytoplasmic proteins. Moreover, confocal image analysis of human 293 cells engineered to express FAC clearly demonstrated that FAC protein is located in both cytoplasmic and nuclear compartments, consistent with data obtained from fractionation of the FA cell lines. Finally, complementation of the FAC defect using retroviral-mediated gene transfer resulted in a substantial increase in nuclear FAC protein. Therefore, while cytoplasmic localization of this protein appears to be functionally important, it may also exert some essential nuclear function.

DNA cross-linker-induced G2/M arrest in group C Fanconi anemia lymphoblasts reflects normal checkpoint function.

Cells from individuals with Fanconi anemia (FA) arrest excessively in the G2/M cell cycle compartment after exposure to low doses of DNA cross-linking agents. The relationship of this abnormality to the fundamental genetic defect in such cells is unknown, but many investigators have speculated that the various FA genes directly regulate cell cycle checkpoints. We tested the hypothesis that the protein encoded by the FA group C complementing gene (FAC) functions to control a cell cycle checkpoint and that cells from group C patients (FA[C]) have abnormalities of cell cycle regulation directly related to the genetic mutation. We found that retroviral transduction of FA(C) lymphoblasts with wild-type FAC cDNA resulted in normalization of the cell cycle response to low-dose mitomycin C (MMC). However, when DNA damage was quantified in terms of cytogenetic damage or cellular cytotoxicity, we found similar degrees of G2/M arrest in response to equitoxic amounts of MMC in FA(C) cells as well as in normal lymphoblasts. Similar results were obtained using isogenic pairs of uncorrected, FAC- or mock-corrected (neo only) FA(C) cell lines. To test the function of other checkpoints we examined the effects of hydroxyurea (HU) and ionizing radiation on cell cycle kinetics of FA(C) and normal lymphoblasts as well as with isogenic pairs of uncorrected, FAC-corrected, or mock-corrected FA(C) cell lines. In all cases the cell cycle response of FA(C) and normal lymphoblasts to these two agents were identical. Based on these studies we conclude that the aberrant G2/M arrest that typifies the response of FA(C) cells to low doses of cross-linking agents does not represent an abnormal cell cycle response but instead represents a normal cellular response to the excessive DNA damage that results in FA(C) cells following exposure to low doses of cross-linking agents.

Transforming growth factor beta 1 inhibits expression of the gene products for steel factor and its receptor (c-kit).

Transforming growth factor beta 1 (TGF-beta 1), a product of marrow stromal cells, inhibits the proliferation and differentiation of hematopoietic progenitor cells within the hematopoietic microenvironment. Steel factor (SF), also a product of marrow stromal cells, is an essential positive regulator of hematopoiesis in vivo. TGF-beta 1 has been shown to repress human and murine leukemic cell and murine lin- bone marrow mononuclear cell expression of the receptor for SF (c-kit). We speculated that TGF-beta 1 might exert its inhibitory effect on hematopoiesis in part by decreasing SF/c-kit interactions. Therefore, we tested the hypothesis that TGF-beta 1 inhibits both stromal cell expression of SF and hematopoietic progenitor cell expression of c-kit. We measured stromal cell expression of SF protein and hematopoietic progenitor cell expression of membrane-bound c-kit before and after exposure to recombinant human TGF-beta 1. Both stromal cell expression of SF protein and hematopoietic progenitor cell expression of c-kit protein were inhibited 50% to 80% by TGF-beta 1. Using Northern blot and ribonuclease protection assays, we determined that TGF-beta 1 repressed stromal cell SF mRNA, but did not alter SF transcript stability. TGF-beta 1 was also found to repress c-kit mRNA in human leukemic myeloblasts as well as in normal lin- hematopoietic progenitor cells. In contrast with its effect on SF mRNA, TGF-beta 1 accelerated the degradation of c-kit mRNA. We conclude that TGF-beta 1 inhibits stromal cell production of SF by repression of SF gene transcription and represses hematopoietic progenitor cell expression of c-kit by decreasing the stability of c-kit transcripts. Taking into account the importance of SF and c-kit in maintaining steady-state hematopoiesis in vivo, the dual effect of TGF-beta 1 on both SF and c-kit gene expression is likely to be one of the major mechanisms by which TGF-beta 1 inhibits hematopoiesis in vivo.

Constitutive expression of steel factor gene by human stromal cells.

Steel factor (SF), the ligand for c-kit, is an essential regulator of normal hematopoiesis, melanogenesis, gametogenesis, and mast-cell growth and development. Hematopoietic stromal cells are important sources of SF, because inactivation of SF in mice results in defects in the support function of hematopoietic stromal cells. To identify specific cells that produce, and factors that govern the expression of the different isoforms of SF in human hematopoiesis, we quantified levels of SF mRNA and membrane-bound protein in human stromal cells before and after exposure to recombinant human interleukin (IL)-1 alpha, a cytokine known to induce the expression of a variety of hematopoietic growth factors. In addition, because stromal cells in longterm bone marrow cultures (LTBMC) are supportive of hematopoietic progenitor cell survival in vitro, while umbilical vein endothelial cells (EC) and diploid fibroblasts (DF) are not, we also sought to test the hypothesis that SF gene expression would differ in cells from LTBMC when compared with EC or DF. Using reverse-transcription polymerase chain reaction amplification (RT-PCR), ribonuclease protection assays (RPA), and Northern blot analysis, SF was found to be constitutively transcribed in EC, DF, and LTBMC. IL-1 alpha neither induced accumulation of SF mRNA nor altered the ratio of exon 6+ to exon 6- transcripts in these stromal cells. By Northern blot analysis, the predominant SF mRNA species was shown to be 5.6 kb; a minor population of 3.6 kb was also found. Low levels of membrane-bound SF protein were found to be constitutively expressed by all three types of stromal cells, and were not regulated by IL-1 alpha. We conclude that the unique capacity of LTBMC to support in vitro hematopoiesis, when compared with EC or DF, cannot be explained on the basis of qualitative or quantitative differences in SF gene expression in these cells.

Amphiregulin messenger RNA is elevated in psoriatic epidermis and gastrointestinal carcinomas.

Amphiregulin (AR) is a heparin-regulated, epidermal growth factor-like growth factor capable of stimulating the proliferation of non-tumorigenic cells while inhibiting cell proliferation in some human tumor cell lines in vitro. In the present study, we have investigated AR mRNA expression in normal, hyperproliferative, and neoplastic human epithelium. Our results demonstrate that, compared with the adjacent uninvolved epithelium, AR mRNA expression is markedly elevated in epidermal biopsies derived from three human psoriatic lesions as well as in biopsies derived from five human colon carcinomas and three human stomach carcinomas. Moreover, analysis of a colon carcinoma by in situ hybridization revealed that AR mRNA is localized to the epithelium.

Inhibition of autonomous human keratinocyte proliferation and amphiregulin mitogenic activity by sulfated polysaccharides.

We previously demonstrated that human keratinocyte cultures proliferate in the absence of polypeptide growth factors (autonomous growth) and that this autonomous growth is blocked by interaction of heparin with a human keratinocyte-derived autocrine factor (KAF) which we identified as amphiregulin (AR). In the present study, we demonstrate that sulfated polysaccharides other than heparin (low and high molecular weight dextran sulfates) also inhibit the AR-mediated autonomous proliferation of human keratinocytes. Furthermore, sulfated polysaccharides such as high and low molecular weight dextran sulfates, heparan sulfate and, to a lesser extent, chondroitin sulfates B and C were also shown to be inhibitors of human keratinocyte-derived AR (k-d AR)-stimulated DNA synthesis in quiescent murine AKR-2B cell cultures. Our results demonstrate that sulfation of polysaccharides is required for AR inhibitory activity, and that several sulfated polysaccharides (other than heparin) can act as inhibitors of AR-mediated autonomous proliferation in human epidermal keratinocytes and as inhibitors of k-d AR-mediated mitogenic activity in AKR-2B cells.

A heparin sulfate-regulated human keratinocyte autocrine factor is similar or identical to amphiregulin.

A novel human keratinocyte-derived autocrine factor (KAF) was purified from conditioned medium by using heparin affinity chromatography as the first step. Purified KAF stimulated the growth of normal human keratinocytes, mouse AKR-2B cells, and a mouse keratinocyte cell line (BALB/MK). Heparin sulfate inhibited KAF mitogenic activity on all cell types tested and inhibited the ability of KAF to compete with epidermal growth factor for cell surface binding. Interestingly, KAF stimulated the growth of BALB/MK cells at high cell density but failed to stimulate these cells at clonal density. Protein microsequencing of the first 20 NH2-terminal amino acid residues of purified KAF revealed identity to the NH2 terminus of human amphiregulin (AR). Northern (RNA) blot analysis with AR-specific cRNA demonstrated that human keratinocytes, as well as mammary epithelial cell cultures, expressed high levels of AR mRNA. In contrast, AR mRNA was not detected in normal human fibroblasts or melanocytes and was present at reduced levels in several mammary tumor cell lines. The mitogenic activity of purified AR was also shown to be inhibited by heparin sulfate, and an AR-specific enzyme-linked immunosorbent assay (ELISA) revealed that KAF and AR are antigenically related. We have previously shown that human keratinocytes can grow in an autocrine manner. Our present study demonstrates that one of the growth factors responsible for this autocrine growth (KAF) is similar or identical to AR and that KAF and AR bioactivity can be negatively regulated by heparin sulfate.

Growth of normal human keratinocytes and fibroblasts in serum-free medium is stimulated by acidic and basic fibroblast growth factor.

Keratinocytes and fibroblasts isolated from human neonatal foreskin can be plated and grown through multiple rounds of division in vitro under defined serum-free conditions. We utilized these growth conditions to examine the mitogenic potential of acidic and basic fibroblast growth factor (aFGF and bFGF) on these cells. Our results demonstrate that both aFGF and bFGF can stimulate the proliferation of keratinocytes and fibroblasts. aFGF is a more potent mitogen than bFGF for keratinocytes. In contrast, bFGF appears to be more potent than aFGF in stimulating the growth of fibroblast cultures. Heparin sulfate (10 micrograms/ml) dramatically inhibited the ability of bFGF to stimulate the proliferation of keratinocytes. In comparison, heparin slightly inhibited the stimulatory effect of aFGF and had no effect on epidermal growth factor (EGF) stimulation in keratinocyte cultures. In fibroblast cultures the addition of heparin enhanced the mitogenic effect of aFGF, had a minimal stimulatory effect on the mitogenic activity of bFGF, and had no effect on EGF-stimulated growth. Our results demonstrate that the proliferation in vitro of two normal cell types found in the skin can be influenced by aFGF and bFGF and demonstrate cell-type specific differences in the responsiveness of fibroblasts and keratinocytes to these growth factors and heparin.

Differential expression of mRNA coding for heparin-binding growth factor type 2 in human cells.

The proliferation of normal human fibroblasts, keratinocytes, and melanocytes in vitro can be controlled by purified polypeptide growth factors and serum. We have studied the cellular expression of the heparin-binding growth factor type 2/basic fibroblast growth factor (HBGF-2/bFGF) gene to determine whether these cell types synthesize mRNA for this mitogen. Our results indicate that normal human fibroblasts synthesize four distinct mRNAs of 7.0, 3.7, 2.2, and 1.5 kilobases, which hybridize to a specific HBGF-2/bFGF cDNA probe. In fibroblasts, the level of all four of these transcripts increases dramatically (more than tenfold) within 4 hours of treatment of quiescent cells with fresh fetal bovine serum. Of the purified growth factors tested, transforming growth factor type-beta also increased HBGF-2/bFGF mRNA abundance, but not to the levels attained by serum treatment. Treatment of fibroblasts with cycloheximide before and during serum treatment blocked the ability of serum to induce the expression of the HBGF-2/bFGF gene. The gene is expressed at low levels in human fibroblasts rapidly growing in serum-free medium and at higher levels in cells rapidly growing in serum-containing medium. In contrast to fibroblasts, mRNA coding for HBGF-2/bFGF is undetectable in proliferating normal human keratinocytes, melanocytes, or mammary epithelial cells. Because keratinocytes and melanocytes proliferate in response to purified HBGF-2/bFGF, our results suggest that HBGF-2/bFGF may mediate the proliferation of epidermal cells through paracrine mechanisms involving stromal fibroblasts. Moreover, we have shown that a human squamous cell carcinoma cell line (SCC-25) expresses mRNA coding for HBGF-2/bFGF, suggesting that the gene may become activated in some carcinomas.