CXCR2 expression during melanoma tumorigenesis controls transcriptional programs that facilitate tumor growth.

BACKGROUND: Though the CXCR2 chemokine receptor is known to play a key role in cancer growth and response to therapy, a direct link between expression of CXCR2 in tumor progenitor cells during induction of tumorigenesis has not been established. METHODS: To characterize the role of CXCR2 during melanoma tumorigenesis, we generated tamoxifen-inducible tyrosinase-promoter driven BrafV600E/Pten-/-/Cxcr2-/- and NRasQ61R/INK4a-/-/Cxcr2-/- melanoma models. In addition, the effects of a CXCR1/CXCR2 antagonist, SX-682, on melanoma tumorigenesis were evaluated in BrafV600E/Pten-/- and NRasQ61R/INK4a-/- mice and in melanoma cell lines. Potential mechanisms by which Cxcr2 affects melanoma tumorigenesis in these murine models were explored using RNAseq, mMCP-counter, ChIPseq, and qRT-PCR; flow cytometry, and reverse phosphoprotein analysis (RPPA). RESULTS: Genetic loss of Cxcr2 or pharmacological inhibition of CXCR1/CXCR2 during melanoma tumor induction resulted in key changes in gene expression that reduced tumor incidence/growth and increased anti-tumor immunity. Interestingly, after Cxcr2 ablation, Tfcp2l1, a key tumor suppressive transcription factor, was the only gene significantly induced with a log2 fold-change greater than 2 in these three different melanoma models. CONCLUSIONS: Here, we provide novel mechanistic insight revealing how loss of Cxcr2 expression/activity in melanoma tumor progenitor cells results in reduced tumor burden and creation of an anti-tumor immune microenvironment. This mechanism entails an increase in expression of the tumor suppressive transcription factor, Tfcp2l1, along with alteration in the expression of genes involved in growth regulation, tumor suppression, stemness, differentiation, and immune modulation. These gene expression changes are coincident with reduction in the activation of key growth regulatory pathways, including AKT and mTOR.

CXCR2 expression during melanoma tumorigenesis controls transcriptional programs that facilitate tumor growth.

Background: Though the CXCR2 chemokine receptor is known to play a key role in cancer growth and response to therapy, a direct link between expression of CXCR2 in tumor progenitor cells during induction of tumorigenesis has not been established. Methods: To characterize the role of CXCR2 during melanoma tumorigenesis, we generated tamoxifen-inducible tyrosinase-promoter driven Braf V600E /Pten -/- /Cxcr2 -/- and NRas Q61R /INK4a -/- /Cxcr2 -/- melanoma models. In addition, the effects of a CXCR1/CXCR2 antagonist, SX-682, on melanoma tumorigenesis were evaluated in Braf V600E /Pten -/- and NRas Q61R /INK4a -/- mice and in melanoma cell lines. Potential mechanisms by which Cxcr2 affects melanoma tumorigenesis in these murine models were explored using RNAseq, mMCP-counter, ChIPseq, and qRT-PCR; flow cytometry, and reverse phosphoprotein analysis (RPPA). Results: Genetic loss of Cxcr2 or pharmacological inhibition of CXCR1/CXCR2 during melanoma tumor induction resulted in key changes in gene expression that reduced tumor incidence/growth and increased anti-tumor immunity. Interestingly, after Cxcr2 ablation, Tfcp2l1 , a key tumor suppressive transcription factor, was the only gene significantly induced with a log 2 fold-change greater than 2 in these three different melanoma models. Conclusions: Here, we provide novel mechanistic insight revealing how loss of Cxcr2 expression/activity in melanoma tumor progenitor cells results in reduced tumor burden and creation of an anti-tumor immune microenvironment. This mechanism entails an increase in expression of the tumor suppressive transcription factor, Tfcp2l1, along with alteration in the expression of genes involved in growth regulation, tumor suppression, stemness, differentiation, and immune modulation. These gene expression changes are coincident with reduction in the activation of key growth regulatory pathways, including AKT and mTOR.

Keratinocyte apoptosis following bone marrow transplantation: evidence for CTL-dependent and -independent pathways.

Acute graft-versus-host disease (GVHD) is a complication of bone marrow transplantation (BMT). The histopathologic features used to diagnose GVHD are nonspecific, and may be secondary to chemotherapy or irradiation given before BMT. The presence of apoptotic keratinocytes or activated CTL may distinguish GVHD from conditioning effects. This study investigated the relationship in BMT recipients between keratinocyte apoptosis and the effects of conditioning regimens or immune-mediated GVHD. Inflammatory cells, apoptotic keratinocytes, and CTL expressing TIA-1 (a molecule associated with the lytic granules of CTL) were quantitated in allogeneic and autologous recipients. Allogeneic recipients could exhibit keratinocyte apoptosis secondary to a combination of conditioning effects and immune-mediated GVHD. In contrast, autologous recipients should show conditioning effects only. 'Capped' TIA-1-positive lymphocytes and apoptotic keratinocytes were much more frequent in the allogeneic group than the autologous group (16.1% of total TIA-1 positive lymphocytes vs 4.5%, P=0.02; and 37.6/mm2 vs 3.9/mm2, P = 0.005, respectively), although there was some overlap in their frequency. Among individual recipients of allogeneic BMT, the number of epidermal lymphocytes or macrophages correlated with the number of apoptotic keratinocytes. A similar, but weaker, correlation was seen between the number of 'capped' TIA-1-positive lymphocytes and apoptotic keratinocytes. No such relationship was seen in autologous recipients. In allogeneic recipients, TIA-1 expressing CTL were seen in intimate contact with apoptotic keratinocytes, some of which also had detectable cytoplasmic TIA-1. No CTL/keratinocyte interactions were identified in autologous recipients. Our results suggest that apoptotic keratinocytes arise in the skin of BMT patients due to both GVHD and conditioning effects, and that the keratinocyte damage in GVHD is mediated by both CTL-dependent and -independent mechanisms. Increased numbers of apoptotic keratinocytes, in the presence of increased epidermal lymphocytes or 'capped' TIA-l-expressing lymphocytes, support a diagnosis of GVHD, but must be interpreted in the context of clinical information and other histopathologic findings.

Implications for the ligase chain reaction in gastroenterology.

The ligase chain reaction (LCR) is a new DNA detection method that uses thermostable ligase to discriminate exquisitely and amplify single base changes in genes of medical interest. This enzyme specifically links two adjacent oligonucleotides when hybridized to a complementary target only when the nucleotides are perfectly base-paired at the junction. Oligonucleotide products are exponentially amplified by thermal cycling of the ligation reaction in the presence of a second set of adjacent oligonucleotides, complementary to the first set and the target. A single-base mismatch prevents ligation and amplification, thus distinguishing a single base mutation from the normal allele. The use of a thermostable ligase allows the enzyme to survive thermal cycling in a fashion analogous to Taq polymerase in the polymerase chain reaction. The assay is compatible with nonradioactive detection and has the potential for automation. Although still in its early stages of development, LCR is expected to find many uses in the field of gastroenterology and in medicine in general. In this review we briefly describe how LCR works and discuss potential areas of application in gastroenterology.

Characterization of steady state, single-turnover, and binding kinetics of the TaqI restriction endonuclease.

The TaqI restriction endonuclease recognizes and cleaves the duplex DNA sequence T decreases CGA. Steady state kinetic analysis with a small oligodeoxyribonucleotide substrate showed that the enzyme obeyed Michaelis-Menten kinetics (Km = 53 nM, kcat = 1.3 min-1 at 50 degrees C and Km = 0.5 nM, kcat = 2.9 min-1 at 60 degrees C). At 0 degree C, the enzyme was completely inactive, while at 15 degrees C, turnover produced nicked substrate as the major product in excess of enzyme indicating dissociation between nicking events. Above 37 degrees C, both strands in the duplex were cleaved prior to dissociation. In contrast to the tight, temperature-dependent binding of substrate, binding of the Mg2+ cofactor was weak (Kd = 2.5 mM) and the same at either 50 degrees C or 60 degrees C. Single-turnover experiments using oligonucleotide substrate showed that hydrolysis of duplex DNA occurred via two independent nicking events, each with a first order rate constant (kst) of 5.8 min-1 at 60 degrees C and 3.5 min-1 at 50 degrees C. The pH dependence of Km (pKa = 9) and kst (pKa = 7) suggests Lys/Arg and His, respectively, as possible amino acids influencing these constants. Moreover, although kst increased significantly with pH, kcat did not, indicating that at least two steps can be rate-controlling in the reaction pathway. Binding of protein to canonical DNA in the presence of Mg2+ at 0 degree C or in the absence of Mg2+ at 50 degrees C was weak (Kd = 2.5 microM or 5,000-fold weaker than the optimal measured Km) and equal to the binding of noncanonical DNA as judged by retention on nitrocellulose. Similar results were seen in gel retardation assays. These results suggest that both Mg2+ and high temperature are required to attain the correct protein conformation to form the tight complex seen in the steady state analysis. In the accompanying paper (Zebala, J. A., Choi, J., Trainor, G. L., and Barany, F. (1992) J. Biol. Chem. 267, 8106-8116), we report how these kinetic constants are altered using substrate analogues and propose a model of functional groups involved in TaqI endonuclease recognition.

DNA recognition of base analogue and chemically modified substrates by the TaqI restriction endonuclease.

It has been proposed that protein-DNA recognition is mediated via specific hydrogen bond, hydrophobic, and/or electrostatic interactions between the protein and DNA surfaces. We have attempted to map and quantitate the energies of these interactions for the TaqI endonuclease by constructing substrates substituted with base or phosphate analogues that either remove or sterically obstruct particular functional groups in the canonical TCGA sequence. The DNA backbone was also modified using a chemical approach (phosphate ethylation) which identified several phosphates in the recognition sequence essential for cleavage. The base analogues, N6-methyl-A, N7-deaza-A, N7-deaza-G, inosine, N4-methyl-C, 5-methyl-C, uracil, 5-bromo-U, and the phosphate analogues, alpha-thio-A, alpha-thio-G, alpha-thio-T, alpha-thio-A, were substituted for their corresponding unmodified counterpart in one strand of the TCGA duplex. The effects of these analogues were monitored by measuring the steady state (Km, kcat) and single-turnover (kst) kinetic constants. Only the N6-methyl-A-substituted DNA, which mimics in vivo methylation, was unreactive while the remaining analogue substitutions exhibited Michaelis-Menten kinetics. In general, the Km was either unchanged or lowered by the analogue substitutions. In contrast, many of the analogues severely reduced kcat, suggesting the modified functional groups served mainly to destabilize the transition state. Single-turnover measurements paralleled the kcat results, pointing to the N7 and N6 of A, the N7 of G, and one of the nonbridging oxygens 3' to T as putative contacts made in achieving the transition state. Substrates with double substitutions displayed simple additivity of delta delta G" implying that these changes behaved independently. The unmodified strand in 10 out of 12 hemisubstituted substrates had a normal kst value suggesting that a particular cleavage center is controlled predominantly by recognition of determinants on the same strand as the scissile bond. These results are discussed in relation to base analogue work from the EcoRI, RsrI, and EcoRV restriction endonucleases.

Correlation between insertion mutant activities and amino acid sequence identities of the TaqI and TthHB8 restriction endonucleases.

A two-codon insertion mutagenesis method has been generalized. Over two dozen insertion mutants throughout the gene encoding TaqI restriction endonuclease were constructed and activity was characterized. All mutants with activity either cleaved or nicked the canonical T decreases CGA recognition sequence. Some insertion mutants created duplication of gene regions, termed Gemini proteins, which still retained activity. The correlation between mutants with poor activity and the regions of shared amino acid identity between the isoschizomeric TaqI and TthHB8I suggests these regions are involved in DNA recognition and/or catalysis.

Cloning and sequencing of genes encoding the TthHB8I restriction and modification enzymes: comparison with the isoschizomeric TaqI enzymes.

Genes encoding the TthHB8I restriction and modification (R-M) system from Thermus thermophilus HB8 (recognition sequence T decreases CGA) were cloned in Escherichia coli. The genes have the same transcriptional orientation, with the last 13 codons of the methyltransferase (MTase) overlapping the first 13 codons of the endonuclease (ENase). Nucleotide sequence analysis of the TthHB8I ENase revealed a single chain of 263 amino acid (aa) residues that share a 77% identity with the corrected isoschizomeric TaqI ENase. Likewise, the Tth MTase (428 aa) shares a 79% identity with the corrected sequence of the TaqI MTase. This high degree of aa conservation suggests a common origin between the Taq and Tth R-M systems. However, codon usage and G+C content for the R-M genes differed markedly from that of other cloned Thermus genes. This suggests that these R-M genes were only recently introduced into the genus Thermus.

Mapping catalytically important regions of an enzyme using two-codon insertion mutagenesis: a case study correlating beta-lactamase mutants with the three-dimensional structure.

Two-codon insertion mutants throughout the beta-lactamase (Bla)-encoding gene were characterized. Second site revertants of various mutants were isolated, mapped and sequenced. The activity of the mutants and the ability to get revertants showed a positive correlation with increasing distance from the active site, based on the three-dimensional structure of Bla. This observation is discussed as it may pertain to the generalized use of two-codon insertion mutagenesis in mapping important catalytic regions in enzymes.