Splenic and immune alterations of the Sparc-null mouse accompany a lack of immune response.

Sparc-null mice have been used as models to assess tumor-host immune cell interactions. However, it is not known if they have a competent immune system. In this study, the immune systems of Sparc wild-type and null mice were compared. Mice were assessed for differences in total body weight, spleen weight and spleen-to-body weight ratios. Spleens were compared with respect to morphology, and Sparc, Ki-67, MOMA-1 and IgM expression. Immune cells in blood, bone marrow and spleen were assessed by blood smears, automated blood panel, and flow cytometry. Additionally, the ability of Sparc-null mice to respond to immune challenge was evaluated using a footpad model. The morphological and immunohistochemical results indicated that Sparc-null spleens had more white pulp, hyperproliferative B cells in the germinal centers, and decreased marginal zones. Sparc-null spleens lacked normal Sparc expression in red and white pulp, marginal zones, endothelial and sinusoidal cells. By flow analysis, B cells were decreased and T cells were increased in the bone marrow. Finally, Sparc-null mice were unable to mount an immune response following footpad lipopolysaccharide challenge. These data confirm that Sparc-null mice have an impaired immune system.

SPARC modulates cell growth, attachment and migration of U87 glioma cells on brain extracellular matrix proteins.

We have identified secreted protein acidic and rich in cysteine (SPARC) as a potential glioma invasion-promoting gene. To determine whether SPARC alters the growth, attachment, or migration of gliomas, we have used U87T2 and doxycycline-regulatable SPARC-transfected clones to examine the effects of SPARC on (1) cell growth, (2) cell cycle progression, (3) cell attachment, and (4) cell migration, using growth curves, flow cytometry, attachment, and migration analyses on different brain ECMs, including collagen IV, laminin, fibronectin, vitronectin, hyaluronic acid, and tenascin. Our data indicate that SPARC delays tumor cell growth in the log phase of the growth curve. The clones secreted different levels of SPARC. The clone secreting the lowest level of SPARC was associated with a higher percentage of cells in G2M, whereas the clones secreting the higher levels of SPARC were associated with a greater percentage of cells in G0/G1. In comparison to the parental U87T2 clone, the SPARC-transfected clones demonstrated increased attachment to collagen, laminin, hyaluronic acid, and tenascin, but not to vitronectin or fibronectin. SPARC-transfected clones also demonstrated altered migration on the different extracellular matrix proteins. The modulation of migration, either positive or negative, was associated with changes in the level of secreted SPARC. These data suggest that SPARC may modulate glioma proliferation and invasion by modulating both the growth and migration of glioma cells.

Increased SPARC expression promotes U87 glioblastoma invasion in vitro.

Our recent studies have focused on identifying invasion-promoting genes that are expressed early in brain tumor progression. We identified and characterized SPARC (secreted protein acidic and rich in cysteine) as a potential candidate. To determine whether increased SPARC expression functionally promotes brain tumor invasion, SPARC was transfected into U87MG glioblastoma cells using the tetracycline-off gene expression system. The parental cell line (U87MG), the parental transactivator-transfected clone (U87T2) and three selected U87T2-SPARC-transfected clones (A2bi, A2b2 and C2a4) were characterized for endogenous and transfected SPARC expression. In comparison to the parental or U87T2 cell lines, the SPARC-transfected clones demonstrated: (1) morphological changes, (2) increased SPARC transcript and protein abundances that were down-regulated by the tetracycline analog doxycycline, (3) perinuclear localization of the transfected SPARC (consistent with reported localization of SPARC in normal cells in culture) and (4) altered adhesion and increased invasion as assessed by the spheroid confrontation assay. These data indicate that increased SPARC expression contributes to U87 glioblastoma tumor invasion in vitro and that these cell lines will serve as useful reagents to investigate the mechanism(s) by which SPARC promotes this phenotype in vitro and in vivo.

SPARC: a signal of astrocytic neoplastic transformation and reactive response in human primary and xenograft gliomas.

In an attempt to identify genetic alterations occurring early in astrocytoma progression, we performed subtractive hybridization between astrocytoma and glioblastoma cDNA libraries. We identified secreted protein acidic and rich in cysteine (SPARC), a protein implicated in cell-matrix interactions, as a gene overexpressed early in progression. Northern blot and immunohistochemical analyses indicated that transcript and protein were both elevated in all tumor specimens (grades II-IV) examined when compared with levels in normal brain. The level of SPARC expression was found to be tumor-dependent rather than grade-related. Immunohistochemically, SPARC protein was found to be overexpressed in 1) cells in the less cellularly dense regions within the tumor mass, 2) histomorphologically neoplastic-looking cells in adjacent normal brain at the tumor/brain interface, 3) neovessel endothelial cells in both the tumor and adjacent normal brain, and 4) reactive astrocytes in normal brain adjacent to tumor. Using a combination of DNA in situ hybridization and protein immunohistochemical analyses of the human/rat xenograft, SPARC expression was observed in the human glioma cells within the tumor mass, and in cells that invaded along vascular basement membranes and individually into the rat brain parenchyma, suggesting it may be an invasion-related gene. While it remains to be determined whether SPARC functionally contributes to tumor cell invasion, these data suggest that the early onset of increased SPARC expression, though complex, may serve as a signal indicative of neoplastic astrocytic transformation and reactive response to tumor-induced stress.

Cathepsin B expression and localization in glioma progression and invasion.

The poor prognosis of human malignant gliomas is due to their invasion and recurrence, the molecular mechanisms of which remain poorly characterized. We have accumulated substantial evidence implicating the cysteine protease cathepsin B in human glioma malignancy. Increases in cathepsin B expression were observed throughout progression. In primary brain tumor tissue, transcript abundance (Northern blot analysis) increased in low-grade astrocytoma to high-grade glioblastoma from 3- to 6-fold, respectively, above normal brain levels. This increase correlated with increases in protein abundance (from + to ) as measured by immunohistochemistry. Furthermore, in glioblastoma cell lines increases in transcript abundance (ranging from 3- to 12-fold) were accompanied by increases in enzyme activity (44-133 nmol/min x mg protein). Altered subcellular localization was observed both immunohistochemically and by indirect immunofluorescence confocal microscopy and was found to correlate with increased grade. In addition, this increase in cathepsin B expression and altered subcellular localization correlated with histomorphological invasion and clinical evidence of invasion as detected by magnetic resonance imaging. These data support the hypothesis that cathepsin B plays a role in human glioma progression and invasion.

Isolation of two contigs of overlapping cosmids derived from human chromosomal band 3p21.1 and identification of 5 new 3p21.1 genes.

Consistent loss of DNA sequences from several regions on the short arm of human chromosome 3 has suggested that multiple tumor suppressor genes reside on chromosome 3p in various types of cancer cells. We have focused our efforts on an analysis of chromosomal band 3p21.1 since aminoacylase-1 (ACY1), which is localized to this band, has been shown to have lower levels of expression in several small cell and non-small cell lung cancer cell lines. Starting with two cosmids within 3p21.1, D3S92 and D3S93, we have isolated two separate contigs of overlapping cosmids within 3p21.1, by screening a library of 5700 chromosome 3-specific cosmid clones. Detailed restriction maps for these two contigs show that they contain multiple clusters of rare cutting restriction endonuclease sites. One contig extends for 100 kb and encompassed both ACY1 and D3S92, and the other extends about 80 kb around the D3S93 locus. Many different restriction fragments derived from these two contigs were found to be evolutionarily conserved and hybridized to distinct message transcripts. These fragments were used to identify homologous cDNAs from an adenogastric cDNA library, and several of these cDNAs were partially sequenced. We have identified five new genes from these two contigs and there is evidence to suggest that several additional genes reside within these cosmid contigs. The genes identified from 3p21.1 were then hybridized to DNA, isolated from a series of lung cancer cell lines and matched normal and tumor DNA from lung cancer patients. No alterations were detected with any of these probes, both at the DNA or RNA levels. A similar analysis with DNA fragments derived from these two genomic regions also failed to detect any alterations.

Physical map of small cell lung cancer deletion region on short arm of human chromosome 3 (3p13-22) based on radiation fusion hybrid analysis.

Deletion of DNA sequences from various regions of the short arm of human chromosome 3 (3p13-14, 3p21, and 3p25) has been observed during the development of a variety of solid tumors, including lung and renal cell carcinomas. In this study we have used a set of radiation fusion hybrids to generate a physical map of chromosome 3p to orient the search for putative tumor suppressor genes. Eighty-six human-hamster radiation fusion hybrids were screened on Southern blots for the retention of 55 human chromosome 3p DNA markers. The high marker density enabled us to identify a set of successively overlapping chromosome fragments in the 3p13-22 area guided by eight markers with previously known order. Twenty-four map intervals were suggested using breakpoints determined by partial fragment overlaps. The final order between the markers derived is consistent with previous information about localizations for 26 of the markers to three larger cytogenetic intervals.

Image analysis of Feulgen-stained NIH/3T3 cells transformed with DNA of 4-nitroquinoline 1-oxide treated human breast epithelial cells.

The nuclear phenotypes of Feulgen-stained NIH/3T3 cells transformed with 4-nitroquinoline 1-oxide (4NQO) treated, human breast epithelial cell (HBEC) DNA were studied by scanning microspectrophotometry and image analysis and compared with data obtained for nontransformed cells and for NIH/3T3 cells under ras oncogene transfecting situations. The Feulgen-DNA content of the individual nuclei (NQ1, NQ2, and NQ3 phenotypes) of the transformed cells was found not to be deeply affected, although presence of chromatin structures resembling double minutes could be verified in part of the metaphases of the transformed cells. On the other hand, the chromatin supraorganization of these cells showed some changes involving increased (NQ2, NQ3) or decreased (NQ1) levels of condensation. The changes in chromatin packing states, however, were of small magnitude compared with those reported for NIH/3T3 cells transfected with a c-H-ras oncogene or an N-ras-containing MCF-7 cell DNA. It was assumed that the transformation of the NIH/3T3 cells is not always necessarily accompanied by high levels of chromatin condensation. The transformation of the NIH/3T3 cells induced by the 4NQO-treated HBEC DNA and particularly the changes in chromatin condensation in these transformed cells could not be attributed merely to a ras activation elicited by the carcinogen. It is suggested that a more complex transforming mechanism is involved, probably owing to the fact that a whole genomic DNA of the 4NQO-treated HBEC has been used for transfection.

Isolation of large numbers of chromosome 3-specific cosmids containing clusters of rare restriction-endonuclease sites.

We tested 519 chromosome 3-specific cosmids for the presence of rare restriction-endonuclease sites in a search for cosmids containing HTF islands. We have identified 49 cosmids (9% of those tested) that contain multiple rare restriction-endonuclease sites. The cosmids were digested with several common cutting restriction endonucleases to liberate small fragments which were tested as unique-sequence chromosome 3-specific hybridization probes and for evolutionary sequence conservation. Unique-sequence hybridization probes isolated from the cosmids were hybridized to a somatic cell hybrid deletion mapping panel to subchromosomally localize the cosmids. Fragments from many of these cosmids demonstrated conservation of sequence through evolution, and these fragments hybridize to distinct transcripts. These cosmids should therefore prove a useful resource for the identification of many chromosome 3-specific genes, in addition to having potential use as linking clones for pulsed-field gel mapping studies.

Localization of 616 human chromosome 3-specific cosmids using a somatic cell hybrid deletion mapping panel.

A total of 5700 human chromosome 3-specific cosmid clones was isolated from a series of cosmid libraries constructed from somatic cell hybrids whose only human component was an entire chromosome 3 or a chromosome 3 containing an interstitial deletion removing 50% of long arm sequences. Several unique sequence chromosome 3-specific hybridization probes were isolated from each of 616 of these cosmids. These probes were then used to localize the cosmids by hybridization to a somatic cell hybrid deletion mapping panel capable of resolving chromosome 3 into nine distinct subregions. All 616 of the cosmids were localized to either the long or short arm of chromosome 3 and 63% of the short arm cosmids were more precisely localized. We have identified a total of 87 cosmids that contain fragments that are evolutionarily conserved. Fragments from these cosmids should prove useful in the identification of new chromosome 3-specific genes as well as in comparative mapping studies. The localized cosmids should provide excellent saturation of human chromosome 3 and facilitate the construction of physical and genetic linkage maps to identify various disease loci including Von Hippel Lindau disease and renal and small cell lung carcinoma.

A 2.5-Mb physical map within 3p21.1 spans the breakpoint associated with Greig cephalopolysyndactyly syndrome.

Numerous investigations suggest that one or more genes residing in the p14 to p21 region of human chromosome 3 are critical to the development of neoplastic diseases such as renal cell carcinoma and small-cell lung cancer (SCLC). This region is additionally involved in several interchromosomal translocations, one of which is associated with the developmental disorder Greig cephalopolysyndactyly syndrome. A series of five loci that map in close proximity to the Greig syndrome breakpoint [t(3;7)(p21.1;p13)] at 3p21.1 have been physically linked by pulsed-field gel analysis over a 2.5-Mb region. The probes include ACY1, cA84 (D3S92), cA199 (D3S93), pHF12-32 (D3S2), and MW-Not153 (D3S332). The Greig 3;7 translocation breakpoint was discovered between clones cA199 and MW-Not153, separated by 825 kb. Further analysis revealed comigration of a rearranged fragment detected by MW-Not153 and a chromosome 7 probe previously shown to be in close proximity to the breakpoint (CRI-R944). This latter probe also detects a rearrangement in a second Greig-associated translocation, (6;7)(q27;p13). The physical map resulting from this analysis orders the markers along the chromosome and identifies several locations for CpG islands, likely associated with genes. Although probe pEFD145.1 (D3S32) has been genetically linked to D3S2 (2 cM), physical linkage to the other five loci could not be demonstrated. One of the linked loci, D3S2, has been widely utilized in the analysis of chromosome 3p loss in several malignant diseases. Since expression of ACY1, a housekeeping gene, is specifically reduced in many cases of SCLC, knowledge of its precise chromosomal position and identification of neighboring putative gene loci should facilitate investigation into the mechanism of this reduction.

Mapping of the gene for human cysteine proteinase inhibitor stefin A, STF1, to chromosome 3cen-q21.

The gene for the human cysteine proteinase inhibitor stefin A (STF1), alias cystatin A, has been mapped to chromosome 3, using the polymerase chain reaction to specifically amplify the human stefin A sequence in human-hamster hybrid DNA. STF1 is further sublocalized to regions between centromere and 3q21 using a deletion mapping panel for this chromosome. This assignment shows that stefin A is not syntenic with cystatin C which has been localized to chromosome 20.

Identification of two cosmids derived from within chromosomal band 3p21.1 that contain clusters of rare restriction sites and evolutionarily conserved sequences.

We have isolated large numbers of human recombinants from a cosmid library constructed from an interspecific (hamster/human) somatic cell hybrid whose only human component is an intact chromosome 3. Unique sequence probes were isolated from these recombinants and were used to localize them along the length of chromosome 3 by hybridization to a somatic cell hybrid deletion panel. We identified two cosmids, cA84 (D3S92) and cA199 (D3S93), derived from within chromosomal band 3p21.1. Both cosmids contained multiple rare restriction sites that were tightly clustered within the cosmids. We have therefore identified, in a region consistently deleted in a variety of lung cancers, two cosmids that may contain genes that are candidates for involvement in lung cancer.

Saturation of human chromosome 3 with unique sequence hybridization probes.

We have generated chromosome 3-specific recombinant libraries in both lambda and cosmid cloning vectors starting with somatic cell hybrids (hamster/human) containing either an intact chromosome 3 or a chromosome 3 with an interstitial deletion removing 75% of long-arm sequences. The libraries contained between 2 X 10(5) and 5 X 10(6) independent recombinants. Approximately 2% of the recombinants in these libraries contain inserts of human DNA. These were identified by hybridizing the recombinants to radioactively labeled total human DNA. Over 2500 recombinants containing human DNA were isolated from these various libraries and DNA was prepared from each of them. This represents 80,000 kb of cloned chromosome 3 sequences. One-third of the DNAs were digested with EcoRI or HindIII, and fragments free of repetitive sequences were radioactively labeled using random hexanucleotide primers and tested as unique sequence hybridization probes. Over 6500 of the fragments were tested and of these 758 were unique sequence probes with minimal or no background hybridization. Their hybridization only to chromosome 3 was verified. These probes, which were derived from 452 independent recombinants, should provide an effective saturation of human chromosome 3.

Overabundance of rare-cutting restriction endonuclease sites in the human genome.

A human chromosome 3-specific cosmid library was constructed from a somatic cell hybrid containing human chromosome 3 as its only human component. This library was screened to identify 230 human recombinants which contained an average insert size of 37 kilobases. DNA prepared from 54 of these cosmids, representing 2000 kilobases of human DNA, was then tested for restriction endonuclease sites for EcoRI, HindIII, KpnI, XhoI, and DraI, as well as those of the rare-cutting restriction endonucleases NotI, SfiI, NruI, MluI, SacII, and BssHII. Sites for the latter enzymes were much more abundant than would be expected from theoretical calculations, reflecting non-random clustering of these sites. This has important implications for the use of these enzymes in the construction of physical maps of chromosomes. Some individual cosmids contained large numbers of rare sites, offering an alternative means of physically mapping chromosomes based upon identifying clusters of rare restriction sites. These clusters appear to be spaced an average of 1000 kb apart.

In vivo stimulation of DNA synthesis and induction of ornithine decarboxylase in rat colon by fatty acid hydroperoxides, autoxidation products of unsaturated fatty acids.

The effect of intrarectal instillation of hydroperoxy and hydroxy fatty acids on colonic DNA synthesis and ornithine decarboxylase activity in male Sprague-Dawley rats was examined. A mixture of hydroperoxy-arachidonic acid isomers was prepared by methylene blue-sensitized photooxygenation. Pure 13-hydroperoxy-9,11-octadecadienoic acid was prepared by the action of soybean lipoxygenase on linoleic acid. Sodium borohydride reduction yielded the respective hydroxy fatty acids. Twelve hr after instillation of solutions of either hydroperoxy or hydroxy fatty acids, at concentrations up to 10 mM, DNA synthesis was increased in a dose-dependent fashion up to 240% above control values. The induction of ornithine decarboxylase occurred over a similar concentration range 3 hr after instillation of oxidized linoleic acid. In this case, the hydroxy acids (49-fold increase at 10 mM), were more stimulatory than the hydroperoxy derivatives (23-fold at 10 mM). Highly purified linoleic and arachidonic acids did not stimulate either activity at concentrations up to 50 mM. These data indicate that autoxidation products of unsaturated fatty acids, likely components of high-fat diets, can evoke proliferative responses in colonic mucosa. These responses may be relevant to the promotional effect of high dietary fat on colon carcinogenesis.