Chemoresistant colorectal cancer cells and cancer stem cells mediate growth and survival of bystander cells.

BACKGROUND: Recent studies suggest that cancer stem cells (CSCs) mediate chemoresistance, but interestingly, only a small percentage of cells in a resistant tumour are CSCs; this suggests that non-CSCs survive by other means. We hypothesised that chemoresistant colorectal cancer (CRC) cells generate soluble factors that enhance survival of chemonaive tumour cells. METHODS: Chemoresistant CRC cells were generated by serial passage in oxaliplatin (Ox cells). Conditioned media (CM) was collected from parental and oxaliplatin-resistant (OxR) cells. CRC cells were treated with CM and growth and survival were assessed. Tumour growth rates were determined in nude mice after cells were treated with CM. Mass spectrometry (MS) identified proteins in CM. Reverse phase protein microarray assays determined signalling effects of CM in parental cells. RESULTS: Oxaliplatin-resistant CM increased survival of chemo-naive cells. CSC CM also increased growth of parental cells. Parental and OxR mixed tumours grew larger than tumours composed of parental or OxR cells alone. Mass spectrometry detected unique survival-promoting factors in OxR CM compared with parental CM. Cells treated with OxR CM demonstrated early phosphorylation of EGFR and MEK1, with later upregulation of total Akt .We identified progranulin as a potential mediator of chemoresistance. CONCLUSION: Chemoresistant tumour cells and CSCs may promote resistance through soluble factors that mediate survival in otherwise chemosensitive tumour cells.

Surfactant impairment after mechanical ventilation with large alveolar surface area changes and effects of positive end-expiratory pressure.

We have assessed the effects of overinflation on surfactant function and composition in rats undergoing ventilation for 20 min with 100% oxygen at a peak inspiratory pressure of 45 cm H2O, with or without PEEP 10 cm H2O (groups 45/10 and 45/0, respectively). Mean tidal volumes were 48.4 (SEM 0.3) ml kg-1 in group 45/0 and 18.3 (0.1) ml kg-1 in group 45/10. Arterial oxygenation in group 45/0 was reduced after 20 min compared with group 45/10 (305 (71) vs 564 (10) mm Hg); maximal compliance of the P-V curve was decreased (2.09 (0.13) vs 4.16 (0.35) ml cm H2O-1 kg-1); total lung volume at a transpulmonary pressure of 5 cm H2O was reduced (6.5 (1.0) vs 18.8 (1.4) ml kg-1) and the Gruenwald index was less (0.22 (0.02) vs 0.40 (0.05)). Bronchoalveolar lavage fluid from the group of animals who underwent ventilation without PEEP had a greater protein concentration (2.18 (0.11) vs 0.76 (0.22) mg ml-1) and a greater minimal surface tension (37.2 (6.3) vs 24.5 (2.8) mN m-1) than in those who underwent ventilation with PEEP. Group 45/0 had an increase in non-active to active total phosphorus compared with nonventilated controls (0.90 (0.16) vs 0.30 (0.07)). We conclude that ventilation in healthy rats with peak inspiratory pressures of 45 cm H2O without PEEP for 20 min caused severe impairment of pulmonary surfactant composition and function which can be prevented by the use of PEEP 10 cm H2O.

Synthesis and in vitro trypanocidal activity of some novel iron chelating agents.

This report describes the syntheses and in vitro trypanocidal activity of a number of iron (III) chelators against epimastigotes of Trypanosoma cruzi. The compounds examined included a number of lipophilic N-alkyl derivatives of 2-ethyl- and 2-methyl-3-hydroxypyrid-4-ones, N,N'-bis(o-hydroxybenzyl)-(+/-)-trans-1,2-diaminocyclohexane, cyclotetrachromotropylene and four commercially available carboxy derivatives of pyridine, pyrazine, and pyarazole. Benznidazole, the drug clinically used in the treatment of Chagas' disease in humans, served as standard. All compounds were screened in vitro against Trypanosoma cruzi epimastigotes at 50 and 100 micrograms/ml for 72 h of exposure. At 100 micrograms/ml dosage, at least 4 compounds exhibited high epimastigote growth inhibition (65-69%) comparable to benznidazole (72%), whereas 9 compounds showed moderate to fair activity (53-64%) in the in vitro assay. At the lower concentration (50 micrograms/ml), the inhibitory activity of the best of these compounds was reduced significantly (39-48%) compared to the standard drug (59%). The activity of all the carboxylic acids remained in the lower range (4-25%). It is hypothesized that the enhanced activity of some of the compounds is due to their increased lipophilicity which enables them to successfully pass through the cellular membrane of Trypanosoma cruzi epimastigotes. The trypanocidal activities of the most effective compounds were significantly reduced when tested in the presence of added ferric ion.

Chelating agent inhibition of Trypanosoma cruzi epimastigotes in vitro.

A number of chelating agents and some of their derivatives are as effective as, or superior to, benznidazole, the compound currently in clinical use, in the suppression of the reproduction of epimastigotes of Trypanosoma cruzi, the protozoa that causes Chagas' disease. All compounds were examined at a culture concentration of 5 micrograms/mL. The most effective compounds included N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine, sodium diethylamine-N-carbodithioate, piperidine-N-carbodithioate and several of its analogs, a number of other carbodithioates with two nonpolar groups on the nitrogen, and tetraethylthiuram disulfide, a prodrug of sodium diethylamine-N-carbodithioate and widely used in the treatment of alcoholism. The introduction of additional ionic or nonionic polar groups on the chelating molecule generally results in a loss of tyrpanocidal activity. Common commercially available chelating agents which exhibited no activity included D-penicillamine, meso-2,3-dimercaptosuccinic acid, and triethylenetetramine tetrahydrochloride. Dose-response data on the culture indicated that some of these compounds exhibited inhibition of Trypanosoma cruzi epimastigotes at concentrations as low as 0.625 microgram/mL. It is proposed that the mechanism of action of these compounds is based on their ability to interfere with the essential metal metabolism at intracellular sites of the epimastigote involving iron, copper, or zinc. The results also indicate that a certain degree of hydrophobicity may be necessary for the groups attached to the literal metal-bonding structure if the compounds are to successfully inhibit the epimastigotes of Trypanosoma cruzi. The development of antiprotozoal drugs which are chelating agents specifically designed to selectively disrupt the essential metal metabolism of Trypanosoma cruzi should furnish a new generation of drugs which can be used in the treatment of Chagas' disease.

Effects of some chelating agents on urinary copper excretion by the rat.

In order to estimate the potential advantages of new chelating agents which can enhance copper excretion in the chronic copper intoxication arising in Wilson's disease, the relative ability of none chelating agents to induce the urinary excretion of copper was compared with that of D-penicillamine (DPA) and triethylenetetramine.2HCl (TRIEN), all given ip at 1 mmol/kg to male Sprague-Dawley rats. The compounds examined were as follows: tris(2-aminoethyl)-amine.3HCl (TREN), tetraethylenepentamine.5HCl (TETREN), pentaethylenehexamine.6HCl (PENTEN), 1,4,7,11-tetraazaundecane.4HCl (TAUD), 1,5,8,12-tetraazadodecane.4HCl (TADD), 1-N-benzyltriethylenetetramine.4HCl (BzTT), 4,7,10,13-tetraazatridecanoic acid.2H2SO4 (TTPA), 1,10-bis(2-pyridylmethyl)-1,4,7,10-tetraazadecane.4HCl (BPTETA), and N,N-bis(2-pyridylmethyl)-4-(aminomethyl)benzoic acid (4ABA). Of these, BzTT, TTPA, and 4ABA are new chelating agents not previously reported. The factors by which these chelating agents enhanced copper excretion over control (untreated) levels were as follows: DPA, 7.2; TREN, 1.6; TRIEN, 4.0; TETREN, 10.1; PENTEN, 7.8; TAUD, 7.8; TADD, 2.6; TTPA, 5.6; BzTT, 1.8; and 4ABA, 5.5. The results indicate that it may well be possible to develop additional chelating agents which are equal or superior to those now used in the treatment of Wilson's disease, as well as structural types whose immunological properties may be significantly different from DPA or TRIEN, the compounds currently used in the clinic for this disorder.

Deletion mutations in the hprt gene of T-lymphocytes as a biomarker for genomic rearrangements important in human cancers.

The DNA sequence of 11 in vivo-arising intragenic deletion junctions occurring in the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene of human T-lymphocytes was determined. These deletions ranged in size from 16 bp to 4057 bp. Extensive homology was not found at the deletion breaksites, indicating that non-homologous recombination was responsible for these deletions. Short regions of homology (1-3 nucleotides) at the deletion termini, which may direct the recombination event, were found in seven of the mutations. Only one mutation had an unaccounted for nucleotide at the junction. V(D)J recombinase recognition sequences, previously identified at other hprt deletion breaksites, were not present. Such features are also found at the deletion and translocation junctions of rearranged oncogenes and suppressor oncogenes. The ability to isolate and molecularly analyze deletion mutations occurring in vivo in peripheral human T-lymphocytes allows the assay of DNA breakage/rejoining events. Such a system may serve as a biomarker of exposure to environmental and occupational agents which may be important in the etiology of cancer.

Multiplex PCR analysis of in vivo-arising deletion mutations in the hprt gene of human T-lymphocytes.

A multiplex polymerase chain reaction (PCR) procedure was adapted for the rapid and efficient evaluation of deletions of the hypoxanthine guanine phosphoribosyltransferase (hprt) gene in human T-lymphocytes. The hprt clonal assay was used to isolate in vivo-arising hprt-deficient T-cells from six healthy males. Mutant frequencies ranged from 9-27 x 10(-6). Simple crude cellular extracts from 223 mutants were analyzed for hprt gene deletion. Sixteen (7.2%) were found to be due to total gene deletion and 22 (9.9%) were due to partial gene deletion. The relatively high frequency of total gene deletions was caused by replicate isolates of a single mutational event as shown by single-strand conformation polymorphism (SSCP) analysis of rearranged T-cell receptor (TCR)-gamma genes. Eighteen of the 22 partial hprt gene deletion mutants were determined to be of independent origin based on a unique hprt mutation or SSCP-TCR -gamma pattern. One-half (9/18) of the partial deletion mutants involved all or part of exon 4 alone, suggesting that this region of the hprt gene is prone to deletion. The small deletions effecting exon 1 (1 mutant), exon 2 (2 mutants), and exon 4 (6 mutants) would not have been detected by conventional Southern blot analysis and may represent a new, previously unrecognized class of mutations. The ready isolation of such intragenic deletions will allow the characterization of breakpoint junctions and may provide insights into the important processes of DNA breakage and rejoining.

Quantification and molecular characterization of hprt mutants of human T-lymphocytes.

Somatic mutations have been implicated as critical early events in carcinogenesis. Point mutations, deletions, and translocation events have been shown to activate oncogenes or inactivate suppressor oncogenes. In human population monitoring, quantitative analysis of mutation events that affect gene function is limited to those genes whose cellular phenotypes can be identified by selection procedures and to those tissues (like blood) that are accessible for analysis. In an effort to determine the frequency and types of mutations that can be detected at the hypoxanthine guanine phosphoribosyltransferase (hprt) gene, we have used the T-cell cloning assay and have developed a strategy to propagate mutants and screen for point mutations and breakage events. Early in the clonal expansion of mutants, 1-2 x 10(4) cells are prepared as a crude cell lysate, and a sample is analyzed using the multiplex polymerase chain reaction (PCR). Those mutants that yield altered DNA fragments are then expanded for Southern blot hybridization, PCR, flanking probe isolation, and DNA sequencing. To date we have found presumed point mutations, intragenic deletions, and deletions that extend outside of the hprt gene. By analyzing mutations in selectable, nonessential gene markers, it should be possible to understand mechanisms of both spontaneous and induced genetic damage. An association of these specific genetic events with human diseases and the evaluation of the ability of environmental chemicals to induce these specific types of mutations will lead to a rational basis for evaluating risks from various chemical exposures.

Large deletions are tolerated at the hprt locus of in vivo derived human T-lymphocytes.

A cloning assay was used to recover hprt- T-lymphocytes from adult human males. Analysis of crude cellular extracts by polymerase chain reactions (PCRs) demonstrated that 7% (16/218) of the hprt mutations were due to total deletion of the hprt gene. 14 of the 16 mutants were examined by PCR for the presence of flanking DNA to determine the extent of the deletions. The deletion mutation in 13 mutants was at least 350 kb with 5 of these deletions being at least 700 kb. The largest deletions were greater than 15 times the size of the hprt gene. Therefore, large deletions are tolerated at the hprt locus of human T-lymphocytes.

Sequence and localization of human NASP: conservation of a Xenopus histone-binding protein.

In this study the sequence and localization of human testicular NASP (nuclear autoantigenic sperm protein) are reported. NASP cDNA contains 2561 nt encoding a protein of 787 amino acids. The open reading frame contains 2446 nt followed by an ochre stop codon (TAA) and 104 nucleotides of untranslated sequence containing a poly(A) addition signal 10 bases upstream of the poly(A) tail. Northern blot analysis of human testis poly(A) mRNA indicates a message of approximately 3.2 kb. Multiple sequence alignment (MSA) analysis of the encoded human NASP amino acid sequence with the sequence for the Xenopus histone-binding protein N1/N2 and the rabbit NASP amino acid sequence demonstrates that the human sequence and the Xenopus sequence have extensive amino acid homology upstream of the rabbit initiation codon. Significantly, there is an 85% identity between the human and the rabbit NASP sequences when the alignment starts at the N-terminal of the rabbit sequence and at amino acid 101 of the human sequence. The nuclear translocation signal found in N1/N2 and rabbit NASP is completely conserved in human NASP. The first histone-binding domain of Xenopus is 70% identical and 90% similar to the human NASP domain. The second histone-binding domain of Xenopus is 48% identical and 71% similar to the human NASP domain. MSA analysis of the three sequences generated an unrooted ancestral tree with two branches, indicating that fewer amino acid changes have occurred between the Xenopus and the human sequences than between the Xenopus and the rabbit sequences. In the human testis, NASP is localized predominantly in primary spermatocytes and round spermatids. Spermatogonia, Sertoli cells, Leydig cells, peritubular cells, and other somatic cells do not stain. Human spermatozoa contain NASP in the acrosomal region. Following the acrosome reaction, some NASP remains in the equatorial and postacrosomal regions. We propose that mammalian testes and sperm contain a histone-binding protein which may play a role in regulating the early events of spermatogenesis.

Analysis of X-ray-induced HPRT mutations in CHO cells: insertion and deletions.

Molecular alterations were examined in the hypoxanthine guanine phosphoribosyltransferase (hprt) gene of 41 independent X-ray-induced thioguanine-resistant (TGR) Chinese hamster ovary (CHO) cell clones. Rapid screening of the clones by multiplex polymerase chain reaction (PCR) for the presence or absence of exons revealed that the causes of the mutant phenotype were total gene deletion (26/41), partial gene deletion (4/41), and an insertion (1/41). No alterations of exon number or sizes were apparent in 10 of the mutants. Southern blot analysis confirmed the deletion data and revealed an additional class of mutants that had a gene disruption but retained all hprt exons (2/41). Therefore, at least 80% of the ionizing radiation-induced mutations were due to mechanisms involving DNA breakage and rejoining. The distribution of deletion sizes suggests that the two DNA breaks required for a deletion are not independent events. A possible mechanism is presented. In addition, the DNA sequence of the insertion mutation was determined. The insertion (229 bp) is coupled with a deletion (31 bp). An imperfect inverted repeat with flanking hprt DNA was identified and may be involved in the insertion event.

V(D)J recombinase-mediated deletion of the hprt gene in T-lymphocytes from adult humans.

The hprt T-cell cloning assay allows the detection of mutations occurring in vivo in the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene of T-lymphocytes. We have shown previously that the illegitimate activity of V(D)J recombinase accounts for about 40% of the hprt mutations in T-lymphocytes of human newborns as measured with umbilical cord blood samples (Fuscoe et al., 1991). This mechanism results in deletion of hprt exons 2 + 3. In this report, we examined a collection of 314 HPRT-deficient clones derived from adult humans for evidence that the mutations were caused by this mechanism by analyzing exons 2 + 3 deletion mutations. DNA sequence analysis of deletion breakpoint junctions showed that 8 of the mutations were the result of V(D)J recombinase activity. The frequency of the recombinase-mediated mutations was similar in the adults and newborns (2-4 x 10(-7). However, since the hprt mutant frequency is about 10-fold higher in the adult than in the newborn, the recombinase-mediated mutations account for only a few percent of the adult mutations. These mutations are likely to have occurred during early development and persist into adulthood. Unregulated expression of V(D)J recombinase activity may be an important mechanism for genomic rearrangements in the genesis of cancer.

V(D)J recombinase-like activity mediates hprt gene deletion in human fetal T-lymphocytes.

Studies from several laboratories worldwide have developed a large database for in vivo hypoxanthine-guanine phosphoribosyltransferase gene mutations in human T-lymphocytes. Sufficient differences have been found thus far between the spectrum for spontaneous mutations in adults and that observed in the fetus to suggest fundamental differences in in vivo mutagenic mechanisms at these two life stages. In adults, only approximately 15% of hypoxanthine-guanine phosphoribosyltransferase mutations have structural alterations on Southern blots, while in the fetus 75% of mutations show alterations of which one-half are deletions of exons 2 and 3. We have now sequenced the breakpoint sites for these specific deletions in 18 mutant lymphocyte clones isolated from 13 normal newborns. Three classes of deletions were found. Each class had the same intron 1 breakpoint but a different intron 3 breakpoint. These mutations have all the signatures of a V(D)J recombinase-mediated event (a 5' consensus heptamer, 3' consensus heptamer and nonamer, nibbling, non-germline-encoded nucleotides, P-nucleotides). At the 3' breakpoint of the most common class (comprising 83% of the mutants) a perfect heptamer can be created by postulating a hairpin loop which could attain a Z-DNA configuration. This feature may indicate recombinase preference for certain DNA structures. These results implicate the V(D)J recombinase in illegitimate events causing mutation in this housekeeping gene during T-cell development. Inactivation of genes involved in the control of growth and differentiation (e.g., tumor suppressor genes) by this mechanism may have important implications for cancer development.

Direct DNA sequencing of PCR products.

Experiments are described which elucidate some of the technical problems associated with the direct sequencing of polymerase chain reaction (PCR) amplified DNA. Sequencing primer purity, labeling methodology, and template preparation were explored. Conditions are presented for the routine sequencing of single- and double-stranded PCR products.

Characterization of a sperm-specific nuclear autoantigenic protein. I. Complete sequence and homology with the Xenopus protein, N1/N2.

In our studies on specific sperm proteins that function in fertilization, an autoantigenic, postacrosomal sperm protein has been found to originate in the testis as a nuclear-associated protein. This nuclear autoantigenic sperm protein (NASP) contains a C-terminal nuclear translocation signal and has structural similarities to the lamins and other nuclear proteins; and its 2.5 kb mRNA is apparently tissue-, but not species-, specific. DNA clones from a rabbit testis cDNA library and a rabbit genomic library were sequenced in order to characterize NASP. The polyadenylated mRNA has 39 bases of 5' untranslated sequence, an open reading frame of 2043 bases encoding 680 amino acids, and a 104 base 3' untranslated region (2,186). The encoded polypeptide has a calculated molecular weight of 73,533 and a pI = 4.06, containing 25% acidic residues. One clone (R1.2) expressing the C-terminal 446 amino acids was used to express a fusion protein. The expressed R1.2/beta-galactosidase fusion protein was found to be autoantigenic. Secondary structure predictions for NASP showed that 69% of the molecule had a high probability of forming alpha-helices and that several alpha-helical regions had a characteristic repeating heptad pattern that in the intermediate filaments and nuclear lamins is involved in coiled-coil interactions with other molecules. In addition to the nuclear translocation signal common to many nuclear proteins, NASP also showed homology with the Xenopus histone-binding protein, N1/N2.