Serum sonic hedgehog (SHH) and interleukin-(IL-6) as dual prognostic biomarkers in progressive metastatic breast cancer.

Serum from one hundred and ten breast cancer patients and thirty healthy female volunteers, were prospectively collected and evaluated for serum levels of Shh and IL-6 using human Shh and IL-6 specific enzyme-linked immunoassays. All patients were regularly monitored for event free survival (EFS) and overall survival (OS). Overall outcome analysis was based on serum Shh and IL-6 levels. In patients with progressive metastatic BC, both serum Shh and IL-6 concentrations were elevated in 44% (29 of 65) and 63% (41 of 65) of patients, respectively, at a statistically significant level [Shh (p = 0.0001) and IL-6 (p = 0.0001)] compared to the low levels in healthy volunteers. Serum levels tended to increase with metastatic progression and lymph node positivity. High serum Shh and IL-6 levels were associated with poor EFS and OS opposite to the negative or lower levels in serum Shh and IL-6. The elevated levels of both serum Shh and IL-6 were mainly observed in BC patients who had a significantly higher risk of early recurrence and bone metastasis, and associated with a worse survival for patients with progressive metastatic BC. Further studies are warranted for validating these biomarkers as prognostic tools in a larger patient cohort and in a longer follow-up study.

Overexpression of sonic hedgehog in the triple negative breast cancer: clinicopathological characteristics of high burden breast cancer patients from Bangladesh.

Dysregulation of Hedgehog (Hh) signaling pathway has been documented in mammary gland development and breast cancer (BC) progression. Despite the remarkable progress in therapeutic interventions, BC related mortality in Bangladesh increased in the last decade. Triple negative breast cancer (TNBC) still presents a critical therapeutic challenge. Thus effective targeted therapy is urgently needed. In this study, we report the clinicopathological characteristics and prognosis of BC patients from Bangladesh. Routine immunohistochemical analysis and high throughput RNA-Seq data from the TCGA library were used to analyze the expression pattern and association of high and low level of Shh expression in a collection of BC patients with a long-term follow-up. High levels of Shh were observed in a subset of BC tumors with poor prognostic pathological features. Higher level of Shh expression correlated with a significantly poorer overall survival of patients compared with patients whose tumors expressed a low level of Shh. These data support the contention that Shh could be a novel biomarker for breast cancer that is involved in mediating the aggressive phenotype of BC. We propose that BC patients exhibiting a higher level of Shh expression, representing a subset of BC patients, would be amenable to Shh targeted therapy.

p16(INK4A) represses the paracrine tumor-promoting effects of breast stromal fibroblasts.

Cancer-associated fibroblasts (CAFs), the most abundant and probably the most active cellular component of breast cancer-associated stroma, promote carcinogenesis through paracrine effects; however, the molecular basis remains elusive. We have shown here that p16(INK4A) expression is reduced in 83% CAFs as compared with their normal adjacent counterparts cancer-free tissues isolated from the same patients. This decrease is mainly due to AUF1-dependent higher turnover of the CDKN2A mRNA in CAFs. Importantly, p16(INK4A) downregulation using specific siRNA activated breast fibroblasts and increased the expression/secretion levels of stromal cell-derived factor 1 (SDF-1) and matrix metalloproteinase (MMP)-2. Consequently, media conditioned with these cells stimulated the proliferation of epithelial cells. Furthermore, the migration/invasion of breast cancer cells was also enhanced in an SDF-1-dependent manner. This effect was mediated through inducing an epithelial-mesenchymal transition state. By contrast, increase in p16(INK4A) level through ectopic expression or AUF1 downregulation, reduced the secreted levels of SDF-1 and MMP-2 and suppressed the pro-carcinogenic effects of CAFs. In addition, p16(INK4A)-defective fibroblasts accelerated breast tumor xenograft formation and growth rate in mice. Importantly, tumors formed in the presence of p16(INK4A)-defective fibroblasts exhibited higher levels of active Akt, Cox-2, MMP-2 and MMP-9, showing their greater aggressiveness as compared with xenografts formed in the presence of p16(INK4A)-proficient fibroblasts. These results provide the first indication that p16(INK4A) downregulation in breast stromal fibroblasts is an important step toward their activation.

Study of anticandidal activity of carvacrol and eugenol in vitro and in vivo.

BACKGROUND/AIMS: The mechanism of the anticandidal action of the major phenolic components of oregano and clove essential oils - carvacrol and eugenol - was studied. This activity was also evaluated for the therapeutic efficacy in the treatment of the experimental oral candidiasis induced by Candida albicans in immunosuppressed rats. METHODS: In vitro, the addition of carvacrol at 0.1% or eugenol at 0.2% during the exponential growth of C. albicans was evaluated. The release of substances absorbing at 280 nm by cells treated with these two components was also measured spectrophotometrically. In vivo, oral candidiasis in immunosuppressed rats was established by inoculating 3 x 10(8) cells of C. albicans with a cotton swab on three alternate days. The number of colony counts was evaluated from the oral cavity of rats treated for eight consecutive days with carvacrol, eugenol or nystatin and compared to untreated controls. RESULTS: Carvacrol and eugenol were fungicidal in exponentially growing C. albicans. Interestingly, this fungicidal effect was accompanied by the release of substances absorbing at 280 nm. In an immunosuppressed rat model of oral candidiasis, carvacrol or eugenol treatment significantly (P < 0.05) reduced the number of colony counts sampled from the oral cavity of rats treated for eight consecutive days compared to untreated control rats. Similar results were obtained with nystatin used as a reference treatment. CONCLUSION: The in vitro results indicated that both carvacrol and eugenol exerted an anticandidal effect by a mechanism implicating an important envelope damage. Their in vivo efficacy on experimental oral candidiasis leads us to consider them as possible antifungal agents.

The Saccharomyces cerevisiae RAD9 cell cycle checkpoint gene is required for optimal repair of UV-induced pyrimidine dimers in both G(1) and G(2)/M phases of the cell cycle.

Cells respond to DNA damage by activating both cellular growth arrest and DNA repair processes. In Saccharomyces cerevesiae the RAD9 gene controls DNA damage-mediated cell cycle arrest that is known to allow efficient repair. To ascertain whether RAD9 plays a role in DNA repair per se, the removal of UV-induced photolesions was assessed in synchronized isogenic normal and rad9 cells using the high resolution primer extension technique. The results show that RAD9 is indeed involved in the removal of photolesions from both the transcribed and the non-transcribed strands of the reporter GAL10 gene, in G(1)- as well as G(2)/M-arrested cells. Interestingly, these data also reveal that in both normal and rad9 mutant, the repair strand bias towards the transcribed stand is more pronounced in G(2)/M- than in G(1)-arrested cells. These data indicate that RAD9 coordinate the cellular response to DNA damage by activating both cell cycle checkpoint and excision repair.

p53 is dispensable for UV-induced cell cycle arrest at late G(1) in mammalian cells.

Genotoxic agents, including gamma-rays and UV light, induce transient arrest at different phases of the cell cycle. These arrests are required for efficient repair of DNA lesions, and employ several factors, including the product of the tumor suppressor gene p53 that plays a central role in the cellular response to DNA damage. p53 protein has a major function in the gamma-ray-induced cell cycle delay in G(1) phase. However, it remains uncertain as to whether p53 is also involved in the UV-mediated G(1) delay. This report provides evidence that p53 is not involved in UV-induced cellular growth arrest in late G(1) phase. This has been demonstrated in HeLa cells synchronized at the G(1)/S border by aphidicolin, followed by UV exposure. Interestingly, the length of this p53-independent G(1) arrest has been shown to be UV dose-dependent. Similar results were also obtained with other p53-deficient cell lines, including human promyelocytic leukemia HL-60 and mouse p53 knock-out cells. As expected, all of these cell lines were defective in gamma-ray-induced cell growth arrest at late G(1). Moreover, it is shown that in addition to cell cycle arrest, HL-60 cells undergo apoptosis in G(1) phase in response to UV light but not to gamma-rays. Together, these findings indicate that p53- compromised cells have a differential response following exposure to ionizing radiation or UV light.

TATA-binding protein promotes the selective formation of UV-induced (6-4)-photoproducts and modulates DNA repair in the TATA box.

DNA-damage formation and repair are coupled to the structure and accessibility of DNA in chromatin. DNA damage may compromise protein binding, thereby affecting function. We have studied the effect of TATA-binding protein (TBP) on damage formation by ultraviolet light and on DNA repair by photolyase and nucleotide excision repair in yeast and in vitro. In vivo, selective and enhanced formation of (6-4)-photoproducts (6-4PPs) was found within the TATA boxes of the active SNR6 and GAL10 genes, engaged in transcription initiation by RNA polymerase III and RNA polymerase II, respectively. Cyclobutane pyrimidine dimers (CPDs) were generated at the edge and outside of the TATA boxes, and in the inactive promoters. The same selective and enhanced 6-4PP formation was observed in a TBP-TATA complex in vitro at sites where crystal structures revealed bent DNA. We conclude that similar DNA distortions occur in vivo when TBP is part of the initiation complexes. Repair analysis by photolyase revealed inhibition of CPD repair at the edge of the TATA box in the active SNR6 promoter in vitro, but not in the GAL10 TATA box or in the inactive SNR6 promoter. Nucleotide excision repair was not inhibited, but preferentially repaired the 6-4PPs. We conclude that TBP can remain bound to damaged promoters and that nucleotide excision repair is the predominant pathway to remove UV damage in active TATA boxes.

Nucleotide excision repair and photolyase preferentially repair the nontranscribed strand of RNA polymerase III-transcribed genes in Saccharomyces cerevisiae.

A high-resolution primer extension technique was used to study the relationships between repair, transcription, and mutagenesis in RNA polymerase III transcribed genes in Saccharomyces cerevisiae. The in vivo repair of UV-induced DNA damage by nucleotide excision repair (NER) and by photoreactivation is shown to be preferential for the nontranscribed strand (NTS) of the SNR6 gene. This is in contrast to RNA polymerase II genes in which the NER is preferential for the transcribed strand (TS). The repair-strand bias observed in SNR6 was abolished by inactivation of transcription in a snr6Delta2 mutant, showing a contribution of RNA polymerase III transcription in this phenomenon. The same strand bias for NER (slow in TS, fast in NTS) was discovered in the SUP4 gene, but only outside of the intragenic promoter element (box A). Unexpectedly, the repair in the transcribed box A was similar on both strands. The strand specificity as well as the repair heterogeneity determined in the transcribed strand of the SUP4 gene, correlate well with the previously reported site- and strand-specific mutagenesis in this gene. These findings present a novel view regarding the relationships between DNA repair, mutagenesis, and transcription.

Two human homologs of Rad23 are functionally interchangeable in complex formation and stimulation of XPC repair activity.

XPC-hHR23B protein complex is specifically involved in nucleotide excision repair (NER) of DNA lesions on transcriptionally inactive sequences as well as the nontranscribed strand of active genes. Here we demonstrate that not only highly purified recombinant hHR23B (rhHR23B) but also a second human homolog of the Saccharomyces cerevisiae Rad23 repair protein, hHR23A, stimulates the in vitro repair activity of recombinant human XPC (rhXPC), revealing functional redundancy between these human Rad23 homologs. Coprecipitation experiments with His-tagged rhHR23 as well as sedimentation velocity analysis showed that both rhHR23 proteins in vitro reconstitute a physical complex with rhXPC. Both complexes were more active than free rhXPC, indicating that complex assembly is required for the stimulation. rhHR23B was shown to stimulate an early stage of NER at or prior to incision. Furthermore, both rhHR23 proteins function in a defined NER system reconstituted with purified proteins, indicating direct involvement of hHR23 proteins in the DNA repair reaction via interaction with XPC.

A novel role for the budding yeast RAD9 checkpoint gene in DNA damage-dependent transcription.

Cells respond to DNA damage by arresting cell cycle progression and activating several DNA repair mechanisms. These responses allow damaged DNA to be repaired efficiently, thus ensuring the maintenance of genetic integrity. In the budding yeast, Saccharomyces cerevisiae, DNA damage leads both to activation of checkpoints at the G1, S and G2 phases of the cell cycle and to a transcriptional response. The G1 and G2 checkpoints have been shown previously to be under the control of the RAD9 gene. We show here that RAD9 is also required for the transcriptional response to DNA damage. Northern blot analysis demonstrated that RAD9 controls the DNA damage-specific induction of a large 'regulon' of repair, replication and recombination genes. This induction is cell-cycle independent as it was observed in asynchronous cultures and cells blocked in G1 or G2/M. RAD9-dependent induction was also observed from isolated damage responsive promoter elements in a lacZ reporter-based plasmid assay. RAD9 cells deficient in the transcriptional response were more sensitive to DNA damage than wild-type cells, even after functional substitution of checkpoints, suggesting that this activation may have an important role in DNA repair. Our findings parallel observations with the Escherichia coli SOS system and suggest the existence of an analogous eukaryotic network coordinating the cellular responses to DNA damage.

Detection of nucleotide excision repair incisions in human fibroblasts by immunostaining for PCNA.

During nucleotide excision repair, damaged DNA is incised on both sides of a lesion and an oligomer containing the damage is excised and replaced by repair DNA synthesis. The latter step is accomplished in vitro by proteins that include the DNA polymerase accessory factor PCNA, which binds to DNA ends to initiate repair synthesis. An increased association of PCNA with nuclei occurs after UV irradiation of nonreplicating DNA in normal human fibroblasts, probably following incision of damaged DNA. This property was used to detect the catalysis of nucleotide excision repair incisions in damaged DNA in vivo, by immunostaining of quiescent human fibroblasts with the widely available PC10 antibody. We summarize here a comprehensive survey of PCNA immunostaining in repair-defective xeroderma pigmentosum (XP) cells in comparison to normal cells. XP-A and XP-G cells were completely defective in staining for PCNA 30 min after UV irradiation. This strongly suggests that XPA and XPG proteins are absolutely required in cells before any incisions can be formed in damaged DNA. XP-B, XP-C, XP-D, and XP-F cells showed an intermediate level of staining for PCNA after UV irradiation, indicative of partial incision capacity in those cells. UV-irradiated XP-E and XP-V cells showed normal PCNA immunostaining levels, consistent with evidence that the corresponding factors are not essential for the incision step of repair. The results provide further evidence for the involvement of PCNA in the repair process in vivo and give an alternative to traditional approaches for measurement of nucleotide excision repair capability.

Mammalian DNA nucleotide excision repair reconstituted with purified protein components.

Nucleotide excision repair is the principal way by which human cells remove UV damage from DNA. Human cell extracts were fractionated to locate active components, including xeroderma pigmentosum (XP) and ERCC factors. The incision reaction was then reconstituted with the purified proteins RPA, XPA, TFIIH (containing XPB and XPD), XPC, UV-DDB, XPG, partially purified ERCC1/XPF complex, and a factor designated IF7. UV-DDB (related to XPE protein) stimulated repair but was not essential. ERCC1- and XPF-correcting activity copurified with an ERCC1-binding polypeptide of 110 kDa that was absent in XP-F cell extract. Complete repair synthesis was achieved by combining these factors with DNA polymerase epsilon, RFC, PCNA, and DNA ligase I. The reconstituted core reaction requires about 30 polypeptides.

Repair of UV-damaged DNA by mammalian cells and Saccharomyces cerevisiae.

Cells use many strategies to repair genomic damage caused by environmental agents and arising from the natural instability of the polynucleotide structure. Nucleotide excision repair is the most versatile DNA repair pathway and is the main defense of mammalian cells against UV-induced DNA damage. Defects in proteins involved in this pathway can lead to inherited disorders (such as xeroderma pigmentosum, Cockayne's syndrome and trichothiodystrophy) that are associated with hypersensitivity to sunlight. Most of the proteins and genes involved in these syndromes have now been identified. Study of UV-sensitive yeast RAD mutants has greatly aided this process and has revealed strong conservation of the components of nucleotide excision repair in eukaryotes. It has recently become clear that some of the proteins involved in the DNA repair process have dual functions and also participate in basal transcription and DNA replication.

Semidominant suppressors of Srs2 helicase mutations of Saccharomyces cerevisiae map in the RAD51 gene, whose sequence predicts a protein with similarities to procaryotic RecA proteins.

Eleven suppressors of the radiation sensitivity of Saccharomyces cerevisiae diploids lacking the Srs2 helicase were analyzed and found to contain codominant mutations in the RAD51 gene known to be involved in recombinational repair and in genetic recombination. These mutant alleles confer an almost complete block in recombinational repair, as does deletion of RAD51, but heterozygous mutant alleles suppress the defects of srs2::LEU2 cells and are semidominant in Srs2+ cells. The results of this study are interpreted to mean that wild-type Rad51 protein binds to single-stranded DNA and that the semidominant mutations do not prevent this binding. The cloning and sequencing of RAD51 indicated that the gene encodes a predicted 400-amino-acid protein with a molecular mass of 43 kDa. Sequence comparisons revealed homologies to domains of Escherichia coli RecA protein predicted to be involved in DNA binding, ATP binding, and ATP hydrolysis. The expression of RAD51, measured with a RAD51-lacZ gene fusion, was found to be UV- and gamma-ray-inducible, with dose-dependent responses.

RADH, a gene of Saccharomyces cerevisiae encoding a putative DNA helicase involved in DNA repair. Characteristics of radH mutants and sequence of the gene.

A new type of radiation-sensitive mutant of S. cerevisiae is described. The recessive radH mutation sensitizes to the lethal effect of UV radiations haploids in the G1 but not in the G2 mitotic phase. Homozygous diploids are as sensitive as G1 haploids. The UV-induced mutagenesis is depressed, while the induction of gene conversion is increased. The mutation is believed to channel the repair of lesions engaged in the mutagenic pathway into a recombination process, successful if the events involve sister-chromatids but lethal if they involve homologous chromosomes. The sequence of the RADH gene reveals that it may code for a DNA helicase, with a Mr of 134 kDa. All the consensus domains of known DNA helicases are present. Besides these consensus regions, strong homologies with the Rep and UvrD helicases of E. coli were found. The RadH putative helicase appears to belong to the set of proteins involved in the error-prone repair mechanism, at least for UV-induced lesions, and could act in coordination with the Rev3 error-prone DNA polymerase.