Evidence for keratinocyte stem cells in vitro: Long term engraftment and persistence of transgene expression from retrovirus-transduced keratinocytes

Epidermis is renewed by a population of stem cells that have been defined in vivo by slow turnover, label retention, position in the epidermis, and enrichment in β1 integrin, and in vitro by clonogenic growth, prolonged serial passage, and rapid adherence to extracellular matrix. The goal of this study is to determine whether clonogenic cells with long-term growth potential in vitro persist in vivo and give rise to a fully differentiated epidermis. Human keratinocytes were genetically labeled in culture by transduction with a retrovirus encoding the lacZ gene and grafted to athymic mice. Analysis of the cultures before grafting showed that 21.1–27.8% of clonogenic cells with the capacity for >30 generations were successfully transduced. In vivo, β-galactosidase (β-gal) positive cells participated in the formation of a fully differentiated epithelium and were detected throughout the 40-week postgraft period, initially as loosely scattered clusters and later as distinct vertical columns. Viable cells recovered from excised grafts were seeded at clonal densities and 23.3–33.3% of the colonies thus formed were β-gal positive. In addition, no evidence of transgene inactivation was obtained: all keratinocyte colonies recovered from grafted tissue that were β-gal negative also lacked the lacZ transgene. These results show that cells with long-term growth properties in vitro do indeed persist in vivo and form a fully differentiated epidermis, thereby exhibiting the properties of stem cells.

Mutagenicity in Escherichia coli of the major DNA adduct derived from the endogenous mutagen malondialdehyde

The spectrum of mutations induced by the naturally occurring DNA adduct pyrimido[1,2-α]purin-10(3H)-one (M1G) was determined by site-specific approaches using M13 vectors replicated in Escherichia coli. M1G was placed at position 6256 in the (−)-strand of M13MB102 by ligating the oligodeoxynucleotide 5′-GGT(M1G)TCCG-3′ into a gapped-duplex derivative of the vector. Unmodified and M1G-modified genomes containing either a cytosine or thymine at position 6256 of the (+)-strand were transformed into repair-proficient and repair-deficient E. coli strains, and base pair substitutions were quantitated by hybridization analysis. Modified genomes containing a cytosine opposite M1G resulted in roughly equal numbers of M1G→A and M1G→T mutations with few M1G→C mutations. The total mutation frequency was ≈1%, which represents a 500-fold increase in mutations compared with unmodified M13MB102. Transformation of modified genomes containing a thymine opposite M1G allowed an estimate to be made of the ability of M1G to block replication. The (−)-strand was replicated >80% of the time in the unadducted genome but only 20% of the time when M1G was present. Correction of the mutation frequency for the strand bias of replication indicated that the actual frequency of mutations induced by M1G was 18%. Experiments using E. coli with different genetic backgrounds indicated that the SOS response enhances the mutagenicity of M1G and that M1G is a substrate for repair by the nucleotide excision repair complex. These studies indicate that M1G, which is present endogenously in DNA of healthy human beings, is a strong block to replication and an efficient premutagenic lesion.

Activation of the c-Jun N-terminal kinase pathway by a novel protein kinase related to human germinal center kinase

The c-Jun N-terminal kinase (JNK), or stress-activated protein kinase plays a crucial role in cellular responses stimulated by environmental stress and proinflammatory cytokines. However, the mechanisms that lead to the activation of the JNK pathway have not been elucidated. We have isolated a cDNA encoding a novel protein kinase that has significant sequence similarities to human germinal center kinase (GCK) and human hematopoietic progenitor kinase 1. The novel GCK-like kinase (GLK) has a nucleotide sequence that encodes an ORF of 885 amino acids with 11 kinase subdomains. Endogenous GLK could be activated by UV radiation and proinflammatory cytokine tumor necrosis factor α. When transiently expressed in 293 cells, GLK specifically activated the JNK, but not the p42/44MAPK/extracellular signal-regulated kinase or p38 kinase signaling pathways. Interestingly, deletion of amino acids 353–835 in the putative C-terminal regulatory region, or mutation of Lys-35 in the putative ATP-binding domain, markedly reduced the ability of GLK to activate JNK. This result indicates that both kinase activity and the C-terminal region of GLK are required for maximal activation of JNK. Furthermore, GLK-induced JNK activation could be inhibited by a dominant-negative mutant of mitogen-activated protein kinase kinase kinase 1 (MEKK1) or mitogen-activated protein kinase kinase 4/SAPK/ERK kinase 1 (SEK1), suggesting that GLK may function upstream of MEKK1 in the JNK signaling pathway.

Molecular origin of cancer: Catechol estrogen-3,4-quinones as endogenous tumor initiators

Cancer is a disease that begins with mutation of critical genes: oncogenes and tumor suppressor genes. Our research on carcinogenic aromatic hydrocarbons indicates that depurinating hydrocarbon–DNA adducts generate oncogenic mutations found in mouse skin papillomas (Proc. Natl. Acad. Sci. USA 92:10422, 1995). These mutations arise by mis-replication of unrepaired apurinic sites derived from the loss of depurinating adducts. This relationship led us to postulate that oxidation of the carcinogenic 4-hydroxy catechol estrogens (CE) of estrone (E1) and estradiol (E2) to catechol estrogen-3,4-quinones (CE-3, 4-Q) results in electrophilic intermediates that covalently bind to DNA to form depurinating adducts. The resultant apurinic sites in critical genes can generate mutations that may initiate various human cancers. The noncarcinogenic 2-hydroxy CE are oxidized to CE-2,3-Q and form only stable DNA adducts. As reported here, the CE-3,4-Q were bound to DNA in vitro to form the depurinating adduct 4-OHE1(E2)-1(α,β)-N7Gua at 59–213 μmol/mol DNA–phosphate whereas the level of stable adducts was 0.1 μmol/mol DNA–phosphate. In female Sprague–Dawley rats treated by intramammillary injection of E2-3,4-Q (200 nmol) at four mammary glands, the mammary tissue contained 2.3 μmol 4-OHE2-1(α,β)-N7Gua/molDNA–phosphate. When 4-OHE1(E2) were activated by horseradish peroxidase, lactoperoxidase, or cytochrome P450, 87–440 μmol of 4-OHE1(E2)-1(α, β)-N7Gua was formed. After treatment with 4-OHE2, rat mammary tissue contained 1.4 μmol of adduct/mol DNA–phosphate. In each case, the level of stable adducts was negligible. These results, complemented by other data, strongly support the hypothesis that CE-3,4-Q are endogenous tumor initiators.

Molecular identification of a novel retrovirus repeatedly isolated from patients with multiple sclerosis

The partial molecular characterization of multiple sclerosis (MS)-associated retrovirus (MSRV), a novel retrovirus previously called LM7, is reported. MSRV has been isolated repeatedly from leptomeningeal, choroid plexus and from Epstein–Barr virus-immortalized B cells of MS patients. A strategy based on reverse transcriptase PCR with RNA-purified extracellular virions yielded an initial pol fragment from which other regions of the retroviral genome were subsequently obtained by sequence extension. MSRV-specific PCR primers amplified a pol region from RNA present at the peak of reverse transcriptase activity, coinciding with extracellular viral particles in sucrose density gradients. The same sequence was detected in noncellular RNA from MS patient plasma and in cerebrospinal fluid from untreated MS patients. MSRV is related to, but distinct from, the endogenous retroviral sequence ERV9. Whether MSRV represents an exogenous retrovirus with closely related endogenous elements or a replication-competent, virion-producing, endogenous provirus is as yet unknown. Further molecular epidemiological studies are required to determine precisely the apparent association of virions containing MSRV RNA with MS.

SMAD3/4-dependent transcriptional activation of the human type VII collagen gene (COL7A1) promoter by transforming growth factor β

The human type VII collagen gene (COL7A1) recently has been identified as an immediate-early response gene for transforming growth factor β (TGF-β)/SMAD signaling pathway. In this study, by using MDA-MB-468 SMAD4−/− breast carcinoma cells, we demonstrate that expression of SMAD4 is an absolute requirement for SMAD-mediated promoter activity. We also demonstrate that the SMAD binding sequence (SBS) representing the TGF-β response element in the region −496/−444 of the COL7A1 promoter functions as an enhancer in the context of a heterologous promoter. Electrophoretic mobility-shift assays with nuclear extracts from COS-1 cells transfected with expression vectors for SMADs 1–5 indicate that SMAD3 forms a complex with a migration similar to that of the endogenous TGF-β-specific complex observed in fibroblast extracts. Electrophoretic mobility-shift assays using recombinant glutathione S-transferase-SMAD fusion proteins indicate that both SMAD4 and C-terminally truncated SMAD3, but not SMAD2, can bind the COL7A1 SBS. Coexpression of SMAD3 and SMAD4 in COS-1 cells leads to the formation of two complexes: a DNA/protein complex containing SMAD3 alone and another slower-migrating complex containing both SMAD3 and SMAD4, the latter complex not being detected in fibroblasts. Maximal transactivation of COL7A1 SBS-driven promoters in either MDA-MB-468 carcinoma cells or fibroblasts requires concomitant overexpression of SMAD3 and SMAD4. These data may represent the first identification of a functional homomeric SMAD3 complex regulating a human gene.

Conservation of the Drosophila lateral inhibition pathway in human lung cancer: A hairy-related protein (HES-1) directly represses achaete-scute homolog-1 expression

The achaete-scute genes encode essential transcription factors in normal Drosophila and vertebrate nervous system development. Human achaete-scute homolog-1 (hASH1) is constitutively expressed in a human lung cancer with neuroendocrine (NE) features, small cell lung cancer (SCLC), and is essential for development of the normal pulmonary NE cells that most resemble this neoplasm. Mechanisms regulating achaete-scute homolog expression outside of Drosophila are presently unclear, either in the context of the developing nervous system or in normal or neoplastic cells with NE features. We now provide evidence that the protein hairy-enhancer-of-split-1 (HES-1) acts in a similar manner as its Drosophila homolog, hairy, to transcriptionally repress achaete-scute expression. HES-1 protein is detected at abundant levels in most non-NE human lung cancer cell lines which lack hASH1 but is virtually absent in hASH1-expressing lung cancer cells. Moreover, induction of HES-1 in a SCLC cell line down-regulates endogenous hASH1 gene expression. The repressive effect of HES-1 is directly mediated by binding of the protein to a class C site in the hASH1 promoter. Thus, a key part of the process that determines neural fate in Drosophila is conserved in human lung cancer cells. Furthermore, modulation of this pathway may underlie the constitutive hASH1 expression seen in NE tumors such as SCLC, the most virulent human lung cancer.

Thrombospondin-2: A potent endogenous inhibitor of tumor growth and angiogenesis

Recent evidence suggests a potential role for thrombospondin-2 (TSP-2), a matricellular glycoprotein, in the regulation of primary angiogenesis. To directly examine the biological effect of TSP-2 expression on tumor growth and angiogenesis, human A431 squamous cell carcinoma cells, which do not express TSP-2, were stably transfected with a murine TSP-2 expression vector or with vector alone. A431 cells expressing TSP-2 did not show an altered growth rate, colony-forming ability, or susceptibility to induction of apoptosis in vitro. However, injection of TSP-2-transfected clones into the dermis of nude mice resulted in pronounced inhibition of tumor growth that was significantly stronger than the inhibition observed in A431 clones stably transfected with a thrombospondin-1 (TSP-1) expression vector, and combined overexpression of TSP-1 and TSP-2 completely prevented tumor formation. Extensive areas of necrosis were observed in TSP-2-expressing tumors, and both the density and the size of tumor vessels were significantly reduced, although tumor cell expression of the major tumor angiogenesis factor, vascular endothelial growth factor, was maintained at high levels. These findings establish TSP-2 as a potent endogenous inhibitor of tumor growth and angiogenesis.

The human granulocyte-macrophage colony- stimulating factor gene is autonomously regulated in vivo by an inducible tissue-specific enhancer

The granulocyte-macrophage colony-stimulating factor (GM-CSF) gene is part of a cytokine gene cluster and is directly linked to a conserved upstream inducible enhancer. Here we examined the in vitro and in vivo functions of the human GM-CSF enhancer and found that it was required for the correctly regulated expression of the GM-CSF gene. An inducible DNase I-hypersensitive site appeared within the enhancer in cell types such as T cells, myeloid cells, and endothelial cells that express GM-CSF, but not in nonexpressing cells. In a panel of transfected cells the human GM-CSF enhancer was activated in a tissue-specific manner in parallel with the endogenous gene. The in vivo function of the enhancer was examined in a transgenic mouse model that also addressed the issue of whether the GM-CSF locus was correctly regulated in isolation from other segments of the cytokine gene cluster. After correction for copy number the mean level of human GM-CSF expression in splenocytes from 11 lines of transgenic mice containing a 10.5-kb human GM-CSF transgene was indistinguishable from mouse GM-CSF expression (99% ± 56% SD). In contrast, a 9.8-kb transgene lacking just the enhancer had a significantly reduced (P = 0.004) and more variable level of activity (29% ± 89% SD). From these studies we conclude that the GM-CSF enhancer is required for the correct copy number-dependent expression of the human GM-CSF gene and that the GM-CSF gene is regulated independently from DNA elements associated with the closely linked IL-3 gene or other members of the cytokine gene cluster.

A 55-kDa protein isolated from human cells shows DNA glycosylase activity toward 3,N4-ethenocytosine and the G/T mismatch

Etheno adducts in DNA arise from multiple endogenous and exogenous sources. Of these adducts we have reported that, 1,N6-ethenoadenine (ɛA) and 3,N4-ethenocytosine (ɛC) are removed from DNA by two separate DNA glycosylases. We later confirmed these results by using a gene knockout mouse lacking alkylpurine-DNA-N-glycosylase, which excises ɛA. The present work is directed toward identifying and purifying the human glycosylase activity releasing ɛC. HeLa cells were subjected to multiple steps of column chromatography, including two ɛC-DNA affinity columns, which resulted in >1,000-fold purification. Isolation and renaturation of the protein from SDS/polyacrylamide gel showed that the ɛC activity resides in a 55-kDa polypeptide. This apparent molecular mass is approximately the same as reported for the human G/T mismatch thymine-DNA glycosylase. This latter activity copurified to the final column step and was present in the isolated protein band having ɛC-DNA glycosylase activity. In addition, oligonucleotides containing ɛC⋅G or G/T(U), could compete for ɛC protein binding, further indicating that the ɛC-DNA glycosylase is specific for both types of substrates in recognition. The same substrate specificity for ɛC also was observed in a recombinant G/T mismatch DNA glycosylase from the thermophilic bacterium, Methanobacterium thermoautotrophicum THF.

Identification of functional domains on human interleukin 10

Interleukin 10 (IL-10) is a recently described natural endogenous immunosuppressive cytokine that has been identified in human, murine, and other organisms. Human IL-10 (hIL-10) has high homology with murine IL-10 (mIL-10) as well as with an Epstein–Barr virus genome product BCRFI. This viral IL-10 (vIL-10) shares a number of activities with hIL-10. IL-10 significantly affects chemokine biology, because human IL-10 inhibits chemokine production and is a specific chemotactic factor for CD8+ T cells. It suppresses the ability of CD4+ T cells, but not CD8+ T cells, to migrate in response to IL-8. A nonapeptide (IT9302) with complete homology to a sequence of hIL-10 located in the C-terminal portion (residues 152–160) of the cytokine was found to possess activities that mimic some of those of hIL-10. These are: (i) inhibition of IL-1β-induced IL-8 production by peripheral blood mononuclear cell, (ii) inhibition of spontaneous IL-8 production by cultured human monocytes, (iii) induction of IL-1 receptor antagonistic protein production by human monocytes, (iv) induction of chemotactic migration of CD8+ human T lymphocytes in vitro, (v) desensitization of human CD8+ T cells resulting in an unresponsiveness toward rhIL-10-induced chemotaxis, (vi) suppression of the chemotactic response of CD4+ T human lymphocytes toward IL-8, (vii) induction of IL-4 production by cultured normal human CD4+ T cells, (viii) down-regulation of tumor necrosis factor-α production by CD8+ T cells, and (ix) inhibition of class II major histocompatibility complex antigen expression on IFN-γ-stimulated human monocytes. Another nonapeptide (IT9403) close to the NH2-terminal part of hIL-10 did not reveal cytokine synthesis inhibitory properties, but proved to be a regulator of mast cell proliferation. In conclusion, we have identified two functional domains of IL-10 exerting different IL-10 like activities, an observation that suggests that relatively small segments of these signal proteins are responsible for particular biological functions.

Somatic Cell Mutants Resistant to Retrovirus Replication: Intracellular Blocks during the Early Stages of Infection

To identify cellular functions involved in the early phase of the retroviral life cycle, somatic cell mutants were isolated after selection for resistance to infection. Rat2 fibroblasts were treated with chemical mutagens, and individual virus-resistant clones were recovered after selection for resistance to infection. Two clones were characterized in detail. Both mutant lines were resistant to infection by both ecotropic and amphotropic murine viruses, as well as by human immunodeficiency virus type 1 pseudotypes. One clone showed a strong block to reverse transcription of the retroviral RNA, including formation of the earliest DNA products. The second clone showed normal levels of viral DNA synthesis but did not allow formation of the circular DNAs normally found in the nucleus. Cell fractionation showed that the viral preintegration complex was present in a form that could not be extracted under conditions that readily extracted the complex from wild-type cells. The results suggest that the DNA was trapped in a nonproductive state and excluded from the nucleus of the infected cell. The properties of these two mutant lines suggest that host gene products play important roles both before and after reverse transcription.

The Endogenous and Cell Cycle-dependent Phosphorylation of tau Protein in Living Cells: Implications for Alzheimer’s Disease

In Alzheimer’s disease the neuronal microtubule-associated protein tau becomes highly phosphorylated, loses its binding properties, and aggregates into paired helical filaments. There is increasing evidence that the events leading to this hyperphosphorylation are related to mitotic mechanisms. Hence, we have analyzed the physiological phosphorylation of endogenous tau protein in metabolically labeled human neuroblastoma cells and in Chinese hamster ovary cells stably transfected with tau. In nonsynchronized cultures the phosphorylation pattern was remarkably similar in both cell lines, suggesting a similar balance of kinases and phosphatases with respect to tau. Using phosphopeptide mapping and sequencing we identified 17 phosphorylation sites comprising 80–90% of the total phosphate incorporated. Most of these are in SP or TP motifs, except S214 and S262. Since phosphorylation of microtubule-associated proteins increases during mitosis, concomitant with increased microtubule dynamics, we analyzed cells mitotically arrested with nocodazole. This revealed that S214 is a prominent phosphorylation site in metaphase, but not in interphase. Phosphorylation of this residue strongly decreases the tau–microtubule interaction in vitro, suppresses microtubule assembly, and may be a key factor in the observed detachment of tau from microtubules during mitosis. Since S214 is also phosphorylated in Alzheimer’s disease tau, our results support the view that reactivation of the cell cycle machinery is involved in tau hyperphosphorylation.

The Human G2 Checkpoint Control Protein hRAD9 Is a Nuclear Phosphoprotein That Forms Complexes with hRAD1 and hHUS1

Eukaryotic cells actively block entry into mitosis in the presence of DNA damage or incompletely replicated DNA. This response is mediated by signal transduction cascades called cell cycle checkpoints. We show here that the human checkpoint control protein hRAD9 physically associates with two other checkpoint control proteins, hRAD1 and hHUS1. Furthermore, hRAD1 and hHUS1 themselves interact, analogously to their fission yeast homologues Rad1 and Hus1. We also show that hRAD9 is present in multiple phosphorylation forms in vivo. These phosphorylated forms are present in tissue culture cells that have not been exposed to exogenous sources of DNA damage, but it remains possible that endogenous damage or naturally occurring replication intermediates cause the observed phosphorylation. Finally, we show that hRAD9 is a nuclear protein, indicating that in this signal transduction pathway, hRAD9 is physically proximal to the upstream (DNA damage) signal rather than to the downstream, cytoplasmic, cell cycle machinery.

Time-dependent vascular regression and permeability changes in established human tumor xenografts induced by an anti-vascular endothelial growth factor/vascular permeability factor antibody

The hyperpermeability of tumor vessels to macromolecules, compared with normal vessels, is presumably due to vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) released by neoplastic and/or host cells. In addition, VEGF/VPF is a potent angiogenic factor. Removal of this growth factor may reduce the permeability and inhibit tumor angiogenesis. To test these hypotheses, we transplanted a human glioblastoma (U87), a human colon adenocarcinoma (LS174T), and a human melanoma (P-MEL) into two locations in immunodeficient mice: the cranial window and the dorsal skinfold chamber. The mice bearing vascularized tumors were treated with a bolus (0.2 ml) of either a neutralizing antibody (A4.6.1) (492 μg/ml) against VEGF/VPF or PBS (control). We found that tumor vascular permeability to albumin in antibody-treated groups was lower than in the matched controls and that the effect of the antibody was time-dependent and influenced by the mode of injection. Tumor vascular permeability did not respond to i.p. injection of the antibody until 4 days posttreatment. However, the permeability was reduced within 6 h after i.v. injection of the same amount of antibody. In addition to the reduction in vascular permeability, the tumor vessels became smaller in diameter and less tortuous after antibody injections and eventually disappeared from the surface after four consecutive treatments in U87 tumors. These results demonstrate that tumor vascular permeability can be reduced by neutralization of endogenous VEGF/VPF and suggest that angiogenesis and the maintenance of integrity of tumor vessels require the presence of VEGF/VPF in the tissue microenvironment. The latter finding reveals a new mechanism of tumor vessel regression—i.e., blocking the interactions between VEGF/VPF and endothelial cells or inhibiting VEGF/VPF synthesis in solid tumors causes dramatic reduction in vessel diameter, which may block the passage of blood elements and thus lead to vascular regression.