Targeted Therapy With PI3K and FGFR Inhibitors on Human Papillomavirus Positive and Negative Tonsillar and Base of Tongue Cancer Lines With and Without Corresponding Mutations.

OBJECTIVES: Human papillomavirus positive (HPV+) tonsillar and base of tongue squamous cell carcinoma (TSCC/BOTSCC), the major subsites of oropharyngeal squamous cell carcinoma (OPSCC) have favorable outcome, but upon relapse, outcome is poor and new therapies needed. Since, phosphatidyl-inositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) and fibroblast-growth-factor-receptor-3 (FGFR3) mutations often occur in such tumors, here, we tested targeted therapy directed to such genes in TSCC/BOTSCC cell lines. We also combined the two types of inhibitors with each other, and cisplatin or docetaxel that are used clinically. METHODS: The HPV+ CU-OP-2, -3, -20, UPCI-SCC-154, and HPV- CU-OP-17 and UT-SCC-60A cell lines were first tested for common PIK3CA/FGFR3 mutations by competitive-allele-specific TaqMan-PCR. They were then treated with the food and drug administration (FDA) approved drugs, alpelisib (BYL719) and erdafitinib (JNJ-42756493) alone and in combination with cisplatin or docetaxel. Viability, proliferation, apoptosis and cytotoxicity responses were thereafter followed by WST-1 assays and the IncuCyte S3 Live® Cell Analysis System. RESULTS: HPV+ CU-OP-2 had a pS249C-FGFR3, and like CU-OP-20, a pE545K-PIK3CA mutation, while no other lines had such mutations. Irrespectively, dose dependent responses to all PI3K/FGFR inhibitors were obtained, and upon combining the inhibitors, positive effects were observed. Cisplatin and docetaxel also induced dose dependent responses, and upon combination with the inhibitors, both positive and neutral effects were found. CONCLUSIONS: The data suggest that FDA approved drugs alpelisib and erdafitinib efficiently inhibit TSCC/BOTSCC cell line growth, especially when combined irrespective of presence of corresponding mutations and should be further explored, for use upon recurrent disease.

In vitro antitumor effects of FGFR and PI3K inhibitors on human papillomavirus positive and negative tonsillar and base of tongue cancer cell lines.

Human papillomavirus positive (HPV+) tonsillar and base of tongue squamous cell carcinoma (TSCC/BOTSCC) have better outcomes than corresponding HPV- negative (HPV-) cancer cases. Our previous study demonstrated that fibroblast growth factor receptor 3 (FGFR3) and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit a (PIK3CA) are often mutated in HPV+ cancer. To investigate whether targeted therapy is an option for TSCC/BOTSCC, two HPV+ and one HPV- TSCC/BOTSCC cell lines were tested for their sensitivity towards FGFR and PI3K inhibitors. The HPV+ cell lines UM-SCC-47 and UPCI-SCC-154, and the HPV- cell line UT-SSC-60A were tested by competitive allele-specific TaqMan-PCR for presence/absence of frequently occurring FGFR3 and PIK3CA mutations. All cells were then treated with FGFR inhibitor AZD4547 and PI3K inhibitors BEZ235 and BKM120 alone, or with AZD4547 and BEZ235 in combination. Viability was analyzed using a WST-1 assay, cytotoxicity tested by a CellTox Green cytotoxicity assay, apoptosis analyzed by a Caspase Glo 3/7 assay and proliferation examined with the xCELLigence system. HPV+ UM-SCC-47 and UPCI-SCC-154 cells, and HPV- UT-SSC-60A cells, did not exhibit any common FGFR3 or PIK3CA mutations, but were all sensitive to FGFR inhibitor AZD4547 and PI3K inhibitors BEZ235 and BKM120. Notably, HPV+ UPCI-SCC-154 cells were more sensitive than the other two cell lines. Furthermore, when AZD4547 and BEZ235 treatment was combined in HPV+ UPCI-SCC-154 and HPV- UT-SSC-60A cells, potentiated combination effects were observed. HPV+ UM-SCC-47 and UPCI-SCC-154 cells, and HPV- UT-SSC-60A cells had no common FGFR3 or PIK3CA mutations, but were sensitive to FGFR inhibitor AZD4547, and PI3K inhibitors BEZ235 and BKM120. Furthermore, the latter two cell lines were particularly sensitive to combinations of AZD4547 and BEZ235.

Analyses of FGFR3 and PIK3CA mutations in neuroblastomas and the effects of the corresponding inhibitors on neuroblastoma cell lines.

Fibroblast growth factor receptor (FGFR)3 and phosphatidylinositol­4,5­bisphosphate 3­kinase, catalytic subunit alpha (PIK3CA) mutations are found in various types of cancer, such as urinary bladder cancer, human papillomavirus­positive tonsillar and base of the tongue squamous cell carcinoma, breast cancer and some childhood sarcomas. Several drugs can target these genes, some of which have been used for the treatment of urinary bladder cancer. Much less is known about childhood cancer. For this reason, the present study investigated the presence of such mutations in neuroblastomas (NBs) and tested NB cell lines for sensitivity to FGFR and phosphoinositide 3­kinase (PI3K) inhibitors. In total, 29 NBs were examined for the presence of the three most common FGFR3 and PIK3CA mutations using a competitive allele­specific TaqMan PCR (CAST­PCR). Furthermore, the SK­N­AS, SK­N­BE(2)­C, SK­N­DZ, SK­N­FI and SK­N­SH NB cell lines (where SK­N­DZ had a deletion of PIK3C2G, none had FGFR mutations according to the Cancer Program's Dependency Map, but some were chemoresistant), were tested for sensitivity to FGFR (AZD4547) and PI3K (BEZ235 and BKM120) inhibitors by viability, cytotoxicity, apoptosis and proliferation assays. CAST­PCR detected one FGFR3 mutation in 1/29 NBs. Following treatment with FGFR and PI3K inhibitors, a decrease in viability and proliferation was observed in the majority, but not all, the cell lines. Following combination treatment with both drugs, the sensitivity of all cell lines was increased. On the whole, the findings of this study demonstrate that FGFR3 and PIK3CA mutations are uncommon in patients with NB. However, certain NB cell lines are rather sensitive to both FGFR and PI3K inhibitors alone, and even more so when the different drugs are used in combination.

Overexpression of FGFR3 in HPV-positive Tonsillar and Base of Tongue Cancer Is Correlated to Outcome.

BACKGROUND/AIM: Human papillomavirus-positive (HPV+) tonsillar and base of tongue squamous cell carcinoma (TSCC/BOTSCC) have better outcome than corresponding HPV- cancers. To better individualize treatment, additional predictive markers are needed. Previously, we have shown that mutated fibroblast growth factor receptor 3 protein (FGFR3) was correlated to poorer prognosis and here FGFR3 expression was further analyzed. PATIENTS AND METHODS: One-hundred-fifteen HPV+TSCC/ BOTSCC biopsies were analyzed for FGFR3 by immunohistochemistry (IHC), and 109/115 were analyzed for FGFR3 mutations by Ion Proton sequencing, or by Competitive Allele-Specific Taqman PCR (CAST-PCR). Disease-free survival (DFS) was then calculated according to FGFR3 IHC expression. RESULTS: CAST-PCR was useful for detecting the three most common FGFR3 mutations. Focusing especially on the 98/115 patients with HPV+TSCC/BOTSCC and wild-type FGFR3, high FGFR3 expression correlated to significantly better 3-year DFS, p=0.043. CONCLUSION: In patients with HPV+TSCC/BOTSCC and wild-type FGFR3, overexpression of FGFR3 was correlated with better DFS.

MicroRNA-155, -185 and -193b as biomarkers in human papillomavirus positive and negative tonsillar and base of tongue squamous cell carcinoma.

OBJECTIVE: Three-year disease-free survival (DFS) is 80% for human papillomavirus (HPV) positive tonsillar and base of tongue cancer (TSCC/BOTSCC) treated with radiotherapy alone, and today's intensified therapy does not improve prognosis. More markers are therefore needed to more accurately identify patients with good prognosis or in need of alternative therapy. Here, microRNAs (miRs) 155, 185 and 193b were examined as potential prognostic markers in TSCC/BOTSCC. MATERIAL AND METHODS: 168 TSCC/BOTSCC patients diagnosed 2000-2013, with known data on HPV-status, CD8+ tumour infiltrating lymphocytes, tumour staging and survival were examined for expression of miR-155, -185 and -193b using Real-Time PCR. Associations between miR expression and patient and tumour characteristics were analysed using univariate testing and multivariate regression. RESULTS: Tumours compared to normal tonsils showed decreased miR-155 and increased miR-193b expression. miR-155 expression was associated with HPV-positivity, low T-stage, high CD8+ TIL counts and improved survival. miR-185 expression was associated with HPV-negativity and a tendency towards decreased survival, while miR-193b expression was associated with higher T-stage, male gender and lower CD8+ TIL counts, but not with outcome. Upon Cox regression, miR-185 was the only miR significantly associated with survival. Combining miR-155 and miR-185 to predict outcome in HPV+ patients yielded an area under curve (AUC) of 71%. CONCLUSION: Increased miR-155 expression was found as a positive predictor of survival, with the effect mainly due to its association with high CD8+ TIL numbers, while miR-185 independently associated with decreased survival. Addition of these miRs to previously validated prognostic biomarkers could improve patient stratification accuracy.

Human papillomavirus (HPV) is absent in branchial cleft cysts of the neck distinguishing them from HPV positive cystic metastasis.

BACKGROUND: Distinguishing branchial cleft cysts (BCCs) from cystic metastases of a human papillomavirus (HPV) positive oropharyngeal squamous cell carcinoma (OPSCC) is challenging. Fine needle aspirates (FNAs) from cystic metastasis may be non-representative, while reactive squamous cells from BCC can be atypic. Based on cytology and with the support of HPV DNA positivity many centers treat cystic metastasis oncological and thus patients are spared neck dissection. To do so safely, one must investigate whether HPV DNA and p16INK4a overexpression is found exclusively in cystic metastases and not in BCC. PATIENTS AND METHODS: DNA was extracted from formalin fixed paraffin embedded (FFPE) surgically resected BCCs from 112 patients diagnosed 2007-2015 at Karolinska University Hospital and amplified by PCR. A multiplex bead-based assay used to detect 27 HPV-types and p16INK4a expression was analyzed by immunohistochemistry (IHC). RESULTS: All 112 BCCs were HPV DNA negative, and of 105 BCCs possible to evaluate for p16INK4a, none overexpressed p16INK4a. CONCLUSIONS: HPV DNA and p16INK4a overexpression were absent in BCCs. Lack of HPV DNA and p16 protein overexpression in BCCs is helpful to discriminate benign BCCs from HPV+ OPSCC metastasis. HPV testing definitely has a role in the diagnostics of cystic masses of the neck.

Protein Expression in Tonsillar and Base of Tongue Cancer and in Relation to Human Papillomavirus (HPV) and Clinical Outcome.

Human papillomavirus (HPV) is a major etiological factor for tonsillar and the base of tongue cancer (TSCC/BOTSCC). HPV-positive and HPV-negative TSCC/BOTSCC present major differences in mutations, mRNA expression and clinical outcome. Earlier protein studies on TSCC/BOTSCC have mainly analyzed individual proteins. Here, the aim was to compare a larger set of cancer and immune related proteins in HPV-positive and HPV-negative TSCC/BOTSCC in relation to normal tissue, presence of HPV, and clinical outcome. Fresh frozen tissue from 42 HPV-positive and 17 HPV-negative TSCC/BOTSCC, and corresponding normal samples, were analyzed for expression of 167 proteins using two Olink multiplex immunoassays. Major differences in protein expression between TSCC/BOTSCC and normal tissue were identified, especially in chemo- and cytokines. Moreover, 34 proteins, mainly immunoregulatory proteins and chemokines, were differently expressed in HPV-positive vs HPV-negative TSCC/BOTSCC. Several proteins were potentially related to clinical outcome for HPV-positive or HPV-negative tumors. For HPV-positive tumors, these were mostly related to angiogenesis and hypoxia. Correlation with clinical outcome of one of these, VEGFA, was validated by immunohistochemistry. Differences in immune related proteins between HPV-positive and HPV-negative TSCC/BOTSCC reflect the stronger activity of the immune defense in the former. Angiogenesis related proteins might serve as potential targets for therapy in HPV-positive TSCC/BOTSCC.

Human Papillomavirus and Potentially Relevant Biomarkers in Tonsillar and Base of Tongue Squamous Cell Carcinoma.

Human papillomavirus (HPV)-positive tonsillar- and base of tongue cancer is increasing epidemically and has much better outcome than corresponding HPV-negative cancer and most other head and neck cancers with around 80% 3-year disease free survival with conventional radiotherapy and surgery. Consequently, most HPV-positive cancer patients may not require the intensified chemoradiotherapy given to many head and neck cancer patients and would, with tapered treatment, avoid several severe side-effects. Moreover, intensified therapy has not improved survival and treatment alternatives are needed. To identify patients eligible for tapered or targeted therapy, additional biomarkers are required. Several studies have, therefore, focused on finding predictive markers, some of which are also potentially targetable. To conclude, better-tailored therapy, either as tapered or targeted, is important for increasing numbers of patients with HPV-positive tonsillar- and base of tongue cancer. This review deals with some of these issues and presents some promising markers.

Targeted sequencing of tonsillar and base of tongue cancer and human papillomavirus positive unknown primary of the head and neck reveals prognostic effects of mutated FGFR3.

BACKGROUND: Human papillomavirus positive (HPV+) tonsillar cancer (TSCC), base of tongue cancer (BOTSCC) and unknown primary cancer of the head and neck (HNCUP) have better outcome than corresponding HPV- cancers. To find predictive markers for response to treatment, and correlations and differences in mutated oncogenes and suppressor genes between HPV+ TSCC/BOTSSCC and HPV+ HNCUP and HPV- TSCC/BOTSCC targeted next-generation sequencing was performed of frequently mutated regions in 50 cancer related genes. PATIENTS AND METHODS: DNA from 348 TSCC/BOTSCC and 20 HNCUP from patients diagnosed 2000-2011, was sequenced by the Ion Proton sequencing platform using the Ion AmpliSeq Cancer Hotspot Panel v2 to identify frequently mutated regions in 50 cancer related genes. Ion Torrent Variant Caller software was used to call variants. RESULTS: 279 HPV+ TSCC/BOTSCC, 46 HPV- TSCC/BOTSCC and 19 HPV+ HNCUP samples qualified for further analysis. Mutations/tumor were fewer in HPV+ TSCC/BOTSCC and HNCUP, compared to HPV- tumors (0.92 vs. 1.32 vs. 1.68). Differences in mutation frequency of TP53 and PIK3CA were found between HPV+ TSCC/BOTSCC and HNCUP and HPV- TSCC/BOTSCC. In HPV+ TSCC/BOTSCC presence of FGFR3 mutations correlated to worse prognosis. Other correlations to survival within the groups were not disclosed. CONCLUSIONS: In HPV+ TSCC/BOTSCC mutation of PIK3CA was most frequently observed, while TP53 mutations dominated in HPV- TSCC/BOTSCC. In HPV+ TSCC/BOTSCC and HNCUP, mutations/tumor were similar in frequency and fewer compared to that in HPV- TSCC/BOTSCC. Notably, FGFR3 mutations in HPV+ TSCC/BOTSCC indicated worse prognosis.

A model using concomitant markers for predicting outcome in human papillomavirus positive oropharyngeal cancer.

OBJECTIVE: Head-neck cancer therapy has become intensified. With radiotherapy alone, 3-year disease-free survival (DFS) is 80% for HPV-positive TSCC/BOTSCC and better for patients with favorable characteristics, suggesting therapy could be tapered for some, decreasing side-effects. Therefore, we built a model to predict progression-free survival for patients with HPV-positive TSCC and BOTSCC. MATERIAL AND METHODS: TSCC/BOTSCC patients treated curatively between 2000 and 2011, with HPV16 DNA/E7 mRNA positive tumors examined for CD8+ TILs, HPV16 mRNA and HLA class I expression were included. Patients were split randomly 65/35 into training and validation sets, and LASSO regression was used to select a model in the training set, the performance of which was evaluated in the validation set. RESULTS: 258 patients with HPV DNA/E7 mRNA positive tumors could be included, 168 and 90 patients in the respective sets. No treatment improved prognosis compared to radiotherapy alone. CD8+ TIL counts and young age were the strongest predictors of survival, followed by T-stage <3 and presence of HPV16 E2 mRNA. The model had an area under curve (AUC) of 76%. A model where the presence of three of four of these markers defined good prognosis captured 56% of non-relapsing patients with a positive predictive value of 98% in the validation set. Furthermore, the model identified 35% of our cohort that was overtreated and could safely have received de-escalated therapy. CONCLUSION: CD8+ TIL counts, age, T-stage and E2 expression could predict progression-free survival, identifying patients eligible for randomized trials with milder treatment, potentially reducing side effects without worsening prognosis.

Effects of irradiation on human leukocyte antigen class I expression in human papillomavirus positive and negative base of tongue and mobile tongue squamous cell carcinoma cell lines.

Human papillomavirus (HPV) infection is a risk factor for oropharyngeal cancer, besides smoking and alcohol. Patients with HPV-positive tumors have a better prognosis than those with HPV-negative tumors. Furthermore, patients with HPV-positive tumors, with high CD8+ tumor infiltrating lymphocyte counts or absent/low human leukocyte antigen (HLA) class I expression have the best outcome. The latter is paradoxical, since HLA class I expression is important for tumor recognition. Below, the hypothesis that radiation therapy increases HLA class I expression was tested. HPV16 positive head and neck cancer cell lines UPCI-SCC-154, UPCI-SCC-090 and UM-SCC-47, and the HPV-negative cancer cell line UT-SCC-14, were treated with 2-10 Gray (Gy) and tested for HLA class I expression, cell cycle changes and apoptosis by flow cytometry. HPV16 E5, E7 and HLA-A mRNA expression was tested by quantitative PCR. A dose of 10 Gy resulted in a tendency of increased HLA class I cell surface expression for all cell lines and reached statistical significance for UPCI-SCC-154 and UPCI-SCC-090. There were, however, no significant changes in HLA-A mRNA expression in any of the cell lines, or HPV16 E5, or E7 mRNA expression for UPCI-SCC-47 and UPCI-SCC-154, while for UPCI-SCC-090 HPV16 E5 mRNA decreased. In all cell lines there was a shift towards G2/M phase and increased apoptosis after irradiation with 10 Gy. To conclude, irradiation with 10 Gy increased HLA class I expression in the HPV-positive cell lines UPCI-SCC-154 and UPCI-SCC-090. A similar tendency was observed for HPV-positive UM-SCC-47 and HPV-negative UT-SCC-14.

Human papillomavirus DNA detection in fine-needle aspirates as indicator of human papillomavirus-positive oropharyngeal squamous cell carcinoma: A prospective study.

BACKGROUND: Human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (SCC) has a better outcome than most head neck squamous cell carcinomas (HNSCCs) and an HPV-positive lymph node metastasis likely has an HPV-positive oropharyngeal SCC origin. Determining HPV-status in cervical lymph nodes by fine-needle aspiration cytology (FNAC) may be useful for diagnosis. METHODS: FNACs from 66 patients with neck masses were prospectively examined for HPV DNA and HPV16 mRNA by a polymerase chain reaction (PCR)-based assay, and the data correlated to diagnosis and HPV-status obtained from histopathological specimens. RESULTS: Aspirates from 17 of 66 patients, later diagnosed with HPV-positive oropharyngeal SCC, were HPV16 DNA-positive. HPV16 mRNA was detected in all cases with extractable RNA. All remaining FNACs, including 18 branchial cleft cysts, were HPV DNA-negative. HPV DNA status in the aspirates showed perfect concordance with corresponding biopsies. CONCLUSION: HPV16 DNA detection in fine-needle aspirations from neck masses is reliable and HPV16 DNA in a metastasis is a strong indicator of an HPV-positive oropharyngeal SCC. © 2016 Wiley Periodicals, Inc. Head Neck 39: 419-426, 2017.

Genome-wide identification of Wig-1 mRNA targets by RIP-Seq analysis.

RNA-binding proteins (RBPs) play important roles in the regulation of gene expression through a variety of post-transcriptional mechanisms. The p53-induced RBP Wig-1 (Zmat3) binds RNA through its zinc finger domains and enhances stability of p53 and N-Myc mRNAs and decreases stability of FAS mRNA. To identify novel Wig-1-bound RNAs, we performed RNA-immunoprecipitation followed by high-throughput sequencing (RIP-Seq) in HCT116 and Saos-2 cells. We identified 286 Wig-1-bound mRNAs common between the two cell lines. Sequence analysis revealed that AU-rich elements (AREs) are highly enriched in the 3'UTR of these Wig-1-bound mRNAs. Network enrichment analysis showed that Wig-1 preferentially binds mRNAs involved in cell cycle regulation. Moreover, we identified a 2D Wig-1 binding motif in HIF1A mRNA. Our findings confirm that Wig-1 is an ARE-BP that regulates cell cycle-related processes and provide a novel view of how Wig-1 may bind mRNA through a putative structural motif. We also significantly extend the repertoire of Wig-1 target mRNAs. Since Wig-1 is a transcriptional target of the tumor suppressor p53, these results have implications for our understanding of p53-dependent stress responses and tumor suppression.

Human papillomavirus is a favourable prognostic factor in cancer of unknown primary in the head and neck region and in hypopharyngeal cancer.

Human papillomavirus (HPV), in addition to smoking and alcohol, is a cause of oropharyngeal squamous cell carcinoma (OPSCC), particularly of the tonsils and base of the tongue (TSCC and BOTSCC, respectively). Moreover, HPV-positive TSCC and BOTSCC are associated with a better outcome compared with their HPV-negative counterparts (80 vs. 40% 3-year disease-free survival rate, respectively) and their incidence has increased in several countries. Recently, accumulating evidence of HPV in a considerable proportion of cancers of unknown primary (CUP) in the head and neck region and in a small proportion of hypopharyngeal SCCs has been reported. Furthermore, HPV-positive tumours, particularly cases with HPV DNA positivity in combination with overexpression of p16, also tend to have a better clinical outcome compared with that of the corresponding HPV-negative tumours. This finding is particularly prominent in HPV-positive CUPs of the head and neck region, where the primary tumour likely originates from the oropharynx. Thus, the determination of HPV status and p16 expression may be of value for the diagnosis and treatment of CUP of the head and neck region and may also be of value for hypopharyngeal cancers in the future. However, for hypopharyngeal cancer as well as other non-OPSCCs, additional studies per subsite on the effect of HPV status on survival are required.

Human papillomavirus DNA and p16(INK4a) expression in hypopharyngeal cancer and in relation to clinical outcome, in Stockholm, Sweden.

OBJECTIVES: Hypopharyngeal cancer is a subset of head neck squamous cell carcinoma (HNSCC) with particularly poor prognosis. Human papillomavirus (HPV) is a risk factor for some HNSCC, and its presence is of prognostic value for certain subsites. However, its influence on survival in hypopharyngeal cancer has not been thoroughly investigated. Here we examine HPV DNA and p16(INK4a) (p16) overexpression in relation to clinical outcome. MATERIALS AND METHODS: Hypopharyngeal tumour biopsies from 82 patients diagnosed 2008-2013 were examined for presence of HPV DNA by a bead-based multiplex assay and for p16 expression by immunohistochemistry, and the obtained data compared to that acquired previously from 109 patients diagnosed 2000-2007 at the same clinic. A survival analysis was then performed on 142 patients (from both studies) treated with curative intent and a 3-year follow-up time. RESULTS: Of the tumour biopsies 3/82 (3.7%) were HPV16 DNA and p16 positive, while 12/82 (14.6%) were p16 positive, equivalent to that in the previous study. Overall 3-year survival was significantly more favourable for patients with HPV16 DNA and p16 positive tumours as compared to survival of the other patients (86% vs. 31%, p=0.0185). A similar but not statistically significant trend was found for disease specific survival. CONCLUSION: HPV DNA and p16 positive hypopharyngeal cancer was rare and had not increased, but had a better clinical outcome as compared to other HPV-unrelated hypopharyngeal cancer. In addition, p16 overexpression was not a suitable surrogate marker for presence of HPV or for prediction of survival in this type of cancer.

The p53 target Wig-1 regulates p53 mRNA stability through an AU-rich element.

The p53 target gene Wig-1 encodes a double-stranded-RNA-binding zinc finger protein. We show here that Wig-1 binds to p53 mRNA and stabilizes it through an AU-rich element (ARE) in the 3' UTR of the p53 mRNA. This effect is mirrored by enhanced p53 protein levels in both unstressed cells and cells exposed to p53-activating stress agents. Thus, the p53 target Wig-1 is a previously undescribed ARE-regulating protein that acts as a positive feedback regulator of p53, with implications both for the steady-state levels of p53 and for the p53 stress response. Our data reveal a previously undescribed link between the tumor suppressor p53 and posttranscriptional gene regulation via AREs in mRNA.

PEG-metronidazole conjugates: synthesis, in vitro and in vivo properties.

Metronidazole (MTZ), a drug used for the treatment of protozoal infections caused by protozoa and anaerobic microorganisms, was conjugated to linear or branched poly(ethylene glycol) of 5,000, 10,000 and 20,000 Da. An ester linkage between polymer and drug was used in the coupling to yield a polymeric prodrug. The modification allowed overcoming the known MTZ solubility problem leading us to obtain a bioconjugate more suitable for parental administration. The conjugates of various molecular weight polymers have been tested in vitro toward chemical degradation and digestive enzymes. It was found that molecular weight and shape of PEG is critical for the prodrugs stability. Good resistance in the stomach acidic media was found and a slow release of the drug in the large intestinal fluid may take place. In vivo studies carried out following i.v. or s.c. administration to mice revealed improved pharmacokinetics properties upon conjugation.