HOXB7: a key factor for tumor-associated angiogenic switch.

We had demonstrated previously a functional bridge between altered homebox (HOX) gene expression and tumor progression through HOXB7 transactivation of basic fibroblast growth factor. Here, we have studied whether HOXB7, in addition to basic fibroblast growth factor, may induce other genes directly or indirectly related to neoangiogenesis and tumor invasion. Parental, beta-galactosidase-transduced, and HOXB7-transduced SkBr3 cell lines were examined for the expression of several growth factors and growth factor receptors involved in the proliferative and angiogenic processes. Vascular endothelial growth factor, melanoma growth-stimulatory activity/growth-related oncogenene alpha, interleukin-8, and angiopoietin-2 were up-regulated by HOXB7 transduction. The exception was angiopoietin-1 expression that was abrogated. Additional analyses included the expression levels of enzymes such as matrix metalloprotease (MMP)-2 and MMP-9 and heparanase, capable of proteolytic degradation of extracellular matrix and basement membranes. Results showed an induction of only MMP-9. The functional implication of such a finding was tested using an in vitro coculture assay in a three-dimensional matrix. A delay of differentiation with persistent nests of proliferating cells was found in endothelial cells cocultured with HOXB7-transduced SkBr3 cells. Tumorigenicity of these cells has been evaluated in vivo. Xenograft into athymic nude mice showed that SkBr3/HOXB7 cells developed tumors in mice, either irradiated or not, whereas parental SkBr3 cells did not show any tumor take unless mice were sublethally irradiated. Comparison of tumor nodules for vascularization by CD-31 and CD-34 immunostaining revealed an increased number of blood vessels in tumors expressing HOXB7. Together, the results indicate HOXB7 as a key factor up-regulating a variety of proangiogenic stimuli. Thus, HOXB7 gene or protein is a target to aim at to inhibit tumor-associated neoangiogenesis, considering the number and the redundancy of proangiogenic molecules that should be targeted one by one to theoretically achieve the same effect.

Enforced expression of HOXB7 promotes hematopoietic stem cell proliferation and myeloid-restricted progenitor differentiation.

Hematopoietic progenitor/stem cells (HPCs/HSCs) purified from human adult peripheral blood (PB) were triggered into cycling, retrovirally transduced with HOXB7 and then functionally assayed in vitro. HPCs were assayed in multi- and unilineage differentiation cultures in either liquid phase or semisolid medium, primitive HPCs in the high proliferative potential colony-forming cell (HPP-CFC) evaluation system and putative HSCs in Dexter type long-term culture (LTC) as LTC initiating cells (LTC-ICs). Control experiments ensured that the exogenous HOXB7 gene was constantly expressed, while the endogenous one was barely or not transcribed. Enforced expression of the gene markedly modulated the proliferation/differentiation program of the entire HSC/HPC population. Enforced HOXB7 expression exerted a potent stimulatory effect on the proliferation of the primitive HPC and putative HSC subsets, assayed as HPP-CFCs and LTC-ICs respectively. While not modifying the total number of HPCs, exogenous HOXB7 induced an increase of the number of granulo-monocytic (GM) HPCs [colony-forming unit GM (CFU-GM) CFU-GM, CFU-G and CFU-M, as evaluated by clonogenic assays] and markedly amplified the progeny of both CFU-G and CFU-M, which showed a sustained proliferation through at least 1-2 months (as evaluated in liquid suspension culture). The prolonged proliferative stimulus induced by HOXB7 transfer into LTC, primitive and GM oriented HPC culture was characterized by persistent proliferation of a discrete population of blast cells and a large pool of differentiated myeloid precursors. Altogether, these results suggest the hypothesis that the proliferative stimulus exerted by exogenous HOXB7 in primitive and GM-oriented HPCs may represent a preleukemic immortalization step. Consistent with the functional role of HOXB7 in the initial ontogenetic phase, these studies indicate that ectopic HOXB7 expression in early HPCs and HSCs from adult PB stimulates their self renewal, sustained proliferation and myeloid differentiation.

Transduction of the SkBr3 breast carcinoma cell line with the HOXB7 gene induces bFGF expression, increases cell proliferation and reduces growth factor dependence.

Several melanomas, carcinomas, glioblastomas and leukemias showed coordinated expression of HOXB7 and bFGF with exception of the SkBr3 mammary carcinoma that was negative for both. Transduction of HOXB7 gene into SkBr3 cells, induced bFGF expression, increased growth rate, independence from serum withdrawal and ability to form colonies in semisolid medium. ELISA assay showed that most of bFGF was associated to cell lysate when cells were cultured at 1% serum whereas in cells kept to 10% serum bFGF was detected both within cell lysate or secreted into cell supernatants. Antisense oligos to bFGF inhibited the growth of cells cultured in 1%, indicating that beside the possible activation of additional genes other than bFGF by HOXB7 transduction, only bFGF induction accounts for the observed results. Moreover, since inhibition of cell proliferation occurred in cells kept in 1% but not 10% serum, a bFGF intracrine loop appears operative in serum starved SkBr3/HOXB7 cells. Also, these results further indicate bFGF as target of HOXB7.

HOXB7 constitutively activates basic fibroblast growth factor in melanomas.

Homeobox (HOX) genes control axial specification during mammalian development and also regulate skin morphogenesis. Although selected HOX genes are variably expressed in leukemias and kidney and colon cancer cell lines, their relationship with the neoplastic phenotype remains unclear. In both normal development and neoplastic transformation, HOX target genes are largely unknown. We investigated the expression and function of HOXB cluster genes in human melanoma. The HOXB7 gene was constitutively expressed in all 25 melanoma cell lines and analyzed under both normal and serum-starved conditions, as well as in in vivo primary and metastatic melanoma cells; conversely, HOXB7 was expressed in proliferating but not quiescent normal melanocytes. Treatment of melanoma cell lines with antisense oligomers targeting HOXB7 mRNA markedly inhibited cell proliferation and specifically abolished expression of basic fibroblast growth factor (bFGF) mRNA. Band shift and cotransfection experiments showed that HOXB7 directly transactivates the hFGF gene through one out of five putative homeodomain binding sites present in its promoter. These novel findings indicate a key role for constitutive HOXB7 expression in melanoma cell proliferation via bFGF. The results also raise the possibility that growth factor genes are critical HOX target genes in other developmental and/or neoplastic cell systems.

Differential expression and functional role of GATA-2, NF-E2, and GATA-1 in normal adult hematopoiesis.

We have explored the expression of the transcription factors GATA-1, GATA-2, and NF-E2 in purified early hematopoietic progenitor cells (HPCs) induced to gradual unilineage erythroid or granulocytic differentiation by growth factor stimulus. GATA-2 mRNA and protein, already expressed in quiescent HPCs, is rapidly induced as early as 3 h after growth factor stimulus, but then declines in advanced erythroid and granulocytic differentiation and maturation. NF-E2 and GATA-1 mRNAs and proteins, though not detected in quiescent HPCs, are gradually induced at 24-48 h in both erythroid and granulocytic culture. Beginning at late differentiation/early maturation stage, both transcription factors are further accumulated in the erythroid pathway, whereas they are suppressed in the granulopoietic series. Similarly, the erythropoietin receptor (EpR) is induced and sustainedly expressed during erythroid differentiation, although beginning at later times (i.e., day 5), whereas it is barely expressed in the granulopoietic pathway. In the first series of functional studies, HPCs were treated with antisense oligomers targeted to transcription factor mRNA: inhibition of GATA-2 expression caused a decreased number of both erythroid and granulocyte-monocytic clones, whereas inhibition of NF-E2 or GATA-1 expression induced a selective impairment of erythroid colony formation. In a second series of functional studies, HPCs treated with retinoic acid were induced to shift from erythroid to granulocytic differentiation (Labbaye et al. 1994. Blood. 83:651-656); this was coupled with abrogation of GATA-1, NF-E2, and EpR expression and conversely enhanced GATA-2 levels. These results indicate the expression and key role of GATA-2 in the early stages of HPC proliferation/differentiation. Conversely, NF-E2 and GATA-1 expression and function are apparently restricted to erythroid differentiation and maturation: their expression precedes that of the EpR, and their function may be in part mediated via the EpR.

Retinoic acid downmodulates erythroid differentiation and GATA1 expression in purified adult-progenitor culture.

All-trans retinoic acid (RA) is an important morphogen in vertebrate development, a normal constituent in human adult blood and is also involved in the control of cell growth and differentiation in acute promyelocytic leukemia. We have examined the effects of RA on normal hematopoiesis by using early hematopoietic progenitor cells (HPC) stringently purified from adult peripheral blood. In clonogenetic fetal calf serum-supplemented (FCS+) or -nonsupplemented (FCS-) culture treated with saturating levels of interleukin-3 (IL-3) granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (Ep) (combined with c-kit ligand in FCS(-)-culture conditions), RA induces a dramatic dose-dependent shift from erythroid to granulomonocytic colony formation, the latter colonies being essentially represented by granulocytic clones. This shift is apparently not caused by a recruitment phenomenon, because in FCS+ culture, the total number of colonies is not significantly modified by RA addition. In FCS- liquid-suspension culture supplemented with saturating Ep level and low-dose IL-3/GM-CSF, adult HPC undergo unilineage erythropoietic differentiation: Here again, treatment with high-dose RA induces a shift from the erythroid to granulocytic differentiation pathway. Studies on RA time-response or pulse treatment in semisolid or liquid culture show that early RA addition is most effective, thus indicating that early but not late HPC are sensitive to its action. We then analyzed the expression of the master GATA1 gene, which encodes a finger transcription factor required for normal erythroid development; addition of RA to HPC stimulated into unilineage erythropoietic differentiation in liquid culture caused a virtually complete inhibition of GATA1 mRNA induction. These results indicate that RA directly inhibits the erythroid differentiation program at the level of early adult HPC, and may lead to a shift from the erythroid to granulocytic differentiation pathway. This phenomenon is correlated with inhibition of GATA1 induction in the early stages of erythropoietic differentiation.

Extrachromosomal human immunodeficiency virus type 1 DNA forms in fresh peripheral blood lymphocytes and in two interleukin-2-independent T cell lines derived from peripheral blood lymphocytes of an asymptomatic seropositive subject.

Two immature T cell lines (FT1 and FT4) were established after in vitro cloning of peripheral blood lymphocytes (PBLs) from an asymptomatic human immunodeficiency virus type 1 (HIV-1) seropositive, human T cell-lymphotropic virus type 1 seronegative homosexual subject. Although derived from a limiting dilution cell cloning assay, these cell lines were not recloned for this study. Their growth was independent of exogenous interleukin-2. Both cell lines were able to form colonies when cloned in agar, but failed to form solid tumours when injected into nude mice. FT lines belong to the very immature T cell lineage as they exhibit rearranged TCR genes but no expression of T cell membrane antigens, including CD2, CD3, CD4, CD6, CD7 and CD8. They also contain an HIV-1 genome that was detected only in an extra-chromosomal DNA form, even after several passages in vitro. The presence of unintegrated viral DNA was also detected by polymerase chain reaction analysis in the same sample of fresh uncultured PBLs. Furthermore, despite the absence of CD4 expression, both T cell lines were susceptible to CD4-independent HIV-1 superinfection (lack of superinfection inhibition in the presence of OKT4A monoclonal antibodies).

Molecular heterogeneity of the 1.0-kb T beta transcript in natural killer and gamma/delta lymphocytes.

The T cell receptor (TcR) beta chain is encoded by a 1.3-kb mRNA which contains variable (V), diversity (D), joining (J) and constant (C) regions. A shorter 1.0-kb transcript with C but no V sequences is found in thymocytes, alpha/beta and gamma/delta T lymphocytes and natural killer cells. The origin, clonal variability and function of this transcript is unknown. We isolated cDNA clones representative of the 1.0-kb mRNA from cDNA libraries of either polyclonal or clonal natural killer and gamma/delta lymphocytes. Ten different types of cDNA clones belonging to three separate groups were identified: (a) "J-C clones" consisting of one of five J beta regions and the corresponding C beta 1 or C beta 2 regions; (b) "D-J-C clones" composed of D beta 1/or D beta 2/J beta rearranged sequences and C beta 1 or C beta 2 sequences; (c) "5'C clones" made up of C beta 1 or C beta 2 regions preceded by the corresponding genomic 5' flanking region. The presence of recombination signal sequences in J-C and D-J-C clones suggests that the first derive from mRNA transcribed from promoters located in the 5' J region and the second from mRNA transcribed from promoters situated in the 5' D region. Nucleotide sequence analysis demonstrated that only three of the ten types of clones isolated had the potential to code a short cytoplasmic T beta protein with a variable D-J beta N terminus. These findings have implications for the mechanisms of T beta germ-line transcription and the function of the T beta transcripts.

Natural killer cells carry the germ-line configuration of the T cell receptor delta chain gene and heterogeneously express six distinct delta transcripts.

Investigations on T cell receptor delta chain (TcR delta) gene expression in freshly isolated normal large granular lymphocytes, natural killer (NK) cell clones and NK bulk cultures revealed six T delta transcripts (3.5, 3.1, 2.2, 2.0, 1.5 and 1.3 kb) which varied in number and relative abundance in the different samples. None of the six known T delta variable regions was expressed and the T delta locus was retained in its germ-line configuration. The origin and significance of these NK-associated T delta transcripts are discussed.