S100A4 is a molecular mediator of endometrial carcinoma invasion

The molecular mechanisms of endometrail cancer invasion are poorly understood. S100A4, a member of the S100 Ca2+-binding protein family, was identified by oligonucleotide microarray qRT-PCR, and IHC, to be highly overexpressed in invasive endometrial carcinomas compared to non-invasive tumors. HEC-1A endometrial cancer cells transfected with S100A4 siRNA had undetectable S100A4 protein, decreased migration and invasion. The mechanism of S100A4 upregulation in endometrial cancer remains unclear. Methylation of the S100A4 gene was detected in benign endometrial glands and grade 1 tumors with no S100A4 expression. In contrast, grade 3 endometrioid tumors with high S100A4 expression showed no methylation of the gene. 5-Aza-2'-deoxycytidine, an inhibitor of DNA methyltransferase, induced the expression of S100A4 in the less invasive EC cell line, KLE, in which the S100A4 gene is hypermethylated and minimally expressed. S100A4 was induced during TGF-β1-triggered cell scattering in HEC-1A cells, in which S100A4 was demethylated. Transfection of HEC-1A cells with S100A4 siRNA significantly reduced the effect of TGF-β1 on basal migration and invasion. Our preliminary data suggested that this upregulation was mediated by the transcription factor Snail. One Snail binding consensus site was found in the region where DNA methylation was closely correlated with S100A4 gene expression. Chromatin immunoprecipitation assay confirmed the binding of Snail to this consensus site in HEC-1A cells. In SPEC2 endometrial cancer cells, loss of Snail leads to repressed S100A4 gene expression. Similar to S100A4, Snail was overexpressed in aggressive endometrial tumors. Our study suggested that the S100A4 gene was demethylated and further upregulated by the TGF-β1 and Snail pathway in invasive endometrial cancer. S100A4 could potentially serve as a good molecular marker for invasiveness and a target for therapeutic intervention for advanced endometrial cancer. ^

Association of exogenous hormonal intake with the risk of development of hepatocellular carcinoma in women

Background. Primary liver cancer, the majority of which is hepatocellular carcinoma, is the third most common cause of mortality from cancer. It has one of the worst prognosis outcomes and an overall 5-year survival of only 5-6%. Hepatocellular carcinoma has been shown to have wide variations in geographic distribution and there is a marked difference in the incidence between different races and gender. Previously low-rate countries, including the US, have shown to have doubled the incidence of HCC during the past two decades. Even though the incidence of HCC is higher in males as compared to females, female hormones, especially estrogens have been postulated to have a role in the development of hepatocellular carcinoma on a molecular level. Despite the frequent usage of oral contraceptive pills (OCP) and previously, hormone replacement therapy (HRT), their role on HCC development has not been studied thoroughly. We aim to examine the association between exogenous hormone intake (oral contraceptives and post-menopausal hormone replacement therapy) and the development of HCC. Methods. This study is part of an ongoing hospital-based case-control study which is conducted at the Department of Gastrointestinal Oncology at The University of Texas M. D. Anderson Cancer Center. From January 2005 up to January 2008, a total of 77 women with pathologically confirmed hepatocellular carcinoma (cases) and 277 healthy women (controls) were included in the investigation. Information about the use of hormonal contraceptives, hormone replacement therapy and risk factors of hepatocellular cancer was collected by personal interview. Univariate and multivariate logistic regression analyses were done to estimate the crude odds ratios (OR) and adjusted odds ratios (AOR). Results. We found statistically significant protective effect for the use of HRT on the development of HCC, AOR=0.42 (95% CI, 0.21, 0.81). The significance was observed for estrogen replacement, AOR=0.43 (95% CI, 0.22, 0.83) and not for progesterone replacement, AOR=0.49 (95% CI, 0.10, 2.35). On the other hand, any hormonal contraceptive use, which encompasses oral contraceptive pills, implants and injections, did not show a statistical significance either in the crude OR=0.58 (95% CI, 0.33, 1.01) or AOR=0.56 (95% CI 0.26, 1.18). Conclusions. As corroborated by previous studies, HRT confers 58% HCC risk reduction among American women. The more important question of the association between hormonal contraceptives and HCC remains controversial. Further studies are warranted to explore the mechanism of the protective effect of HRT and the relationship between hormonal contraception and HCC.^

Estrogen-induced gene expression and patient survival in high-grade serous carcinoma of the ovary

Background. Assessment of estrogen receptor (ER) expression has inconsistent utility as a prognostic marker in epithelial ovarian carcinoma. In breast and endometrial cancers, the use of estrogen-induced gene panels, rather than ER expression alone, has shown improved prognostic capability. Specifically, over-expression of estrogen-induced genes in these tumors is associated with a better prognosis and signifies estrogen sensitivity that can be exploited with hormone antagonizing agents. It was therefore hypothesized that estrogen-induced gene expression in ovarian carcinoma would successfully predict outcomes and differentiate between tumors of varying estrogen sensitivities. Methods. Two hundred nineteen (219) patients with ovarian cancer who underwent surgery at M. D. Anderson between 2004 and 2007 were identified. Of these, eighty-three (83) patients were selected for inclusion because they had advanced stage, high-grade serous carcinoma of the ovary or peritoneum, had not received neoadjuvant chemotherapy, and had readily available frozen tissue for study. All patients had also received adjuvant treatment with platinum and taxane agents. The expression of seven genes known to be induced by estrogen in the female reproductive tract (EIG121, sFRP1, sFRP4, RALDH2, PR, IGF-1, and ER) was measured using qRT-PCR. Unsupervised cluster analyses of multiple gene permutations were used to categorize patients as high or low estrogen-induced gene expressors. QPCR gene expression results were then compared to ER and PR immunohistochemical (IHC) expression. Cox proportional hazards models were used to evaluate the effects of both individual genes and selected gene clusters on patient survival. Results. Median follow-up time was 38.7 months (range 1-68 months). In a multivariate model, overall survival was predicted by sFRP1 expression (HR 1.10 [1.02-1.19], p=0.01) and EIG121 expression (HR 1.28 [1.10-1.49], p<0.01). A cluster defined by EIG121 and ER was further examined because that combination appeared to reasonably segregate tumors into distinct groups of high and low estrogen-induced gene expressors. Shorter overall survival was associated with high estrogen-induced gene expressors (HR 2.84 [1.11-7.30], p=0.03), even after adjustment for race, age, body mass index, and residual disease at debulking. No difference in IHC ER or PR expression was noted between gene clusters. Conclusion. In sharp contrast to breast and endometrial cancers, high estrogen-induced gene expression predicts shorter overall survival in patients with high-grade serous ovarian carcinoma. An estrogen-induced gene biomarker panel may have utility as prognostic indicator and may be useful to guide management with estrogen antagonists in this population.^

Regulation of squamous cell differentiation in human head and neck carcinoma cells by retinoids

Retinoids have been found to be effective in the prevention of premalignant lesions and second primary cancers in the upper aerodigestive tract. Further development of retinoids for prevention and therapy of head and neck squamous cell carcinoma (HNSCC) requires a better understanding of their mechanism of action on the growth and differentiation of such cells. I have chosen to employ cultured HNSCC cell lines as a model system for investigating the mechanism underlying the effects of retinoids. These cells are useful because all-trans retinoic acid (ATRA) inhibits their proliferation. Furthermore, two HNSCC cell lines were found to express three squamous differentiation (SqD) markers characteristic of normal keratinocytes and ATRA suppressed the expression of these markers as reported for normal keratinocytes. It is thought that nuclear retinoic acid receptors (RARs and RXRs), which act as DNA-binding transcription modulating factors, mediate the effects of retinoids on the growth and differentiation of normal and tumor cells. I found that all four cell lines examined expressed RAR-$\alpha ,$ RAR-$\tau ,$ and RXR-$\alpha$ and three of four expressed RAR-$\beta .$ ATRA treatment increased the level of RAR-$\alpha ,$ -$\beta ,$ and -$\tau$ in four cell lines. Two HNSCC cell lines that exhibited a progressive increase in the expression of SqD markers during growth in culture also showed a concurrent decrease in RAR-$\beta$ level. Moreover, increasing concentrations of RA suppressed the SqD marker while inducing RAR-$\beta$ mRNA. Several synthetic retinoids which exhibit a preference for binding to specific nuclear RARs showed a differential ability to inhibit cell proliferation, transactivate transcription of the reporter genes (CAT and luciferase) from the RA response element (RARE) of the RAR-$\beta$ gene, and induce RAR-$\beta$ expression. Those retinoids that were effective inducers of RAR-$\beta$ also suppressed SqD effectively, indicating an inverse relationship exists between the expression of RAR-$\beta$ and SqD. This inverse relationship suggests a role for RAR-$\beta$ in the suppression of SqD. ^

Molecular epidemiological studies on genetic predispositions to squamous cell carcinoma of the head and neck

Two molecular epidemiological studies were conducted to examine associations between genetic variation and risk of squamous cell carcinoma of the head and neck (SCCHN). In the first study, we hypothesized that genetic variation in p53 response elements (REs) may play roles in the etiology of SCCHN. We selected and genotyped five polymorphic p53 REs as well as a most frequently studied p53 codon 72 (Arg72Pro, rs1042522) polymorphism in 1,100 non-Hispanic White SCCHN patients and 1,122 age-and sex-matched cancer-free controls recruited at The University of Texas M. D. Anderson Cancer Center. In multivariate logistic regression analysis with adjustment for age, sex, smoking and drinking status, marital status and education level, we observed that the EOMES rs3806624 CC genotype had a significant effect of protection against SCCHN risk (adjusted odds ratio= 0.79, 95% confidence interval =0.64–0.98), compared with the -838TT+CT genotypes. Moreover, a significantly increased risk associated with the combined genotypes of p53 codon 72CC and EOMES -838TT+CT was observed, especially in the subgroup of non-oropharyneal cancer patients. The values of false-positive report probability were also calculated for significant findings. In the second study, we assessed the association between SCCHN risk and four potential regulatory single nucleotide polymorphisms (SNPs) of DEC1 (deleted in esophageal cancer 1) gene, a candidate tumor suppressor gene for esophageal cancer. After adjustment for age, sex, and smoking and drinking status, the variant -606CC (i.e., -249CC) homozygotes had a significantly reduced SCCHN risk (adjusted odds ratio = 0.71, 95% confidence interval = 0.52–0.99), compared with the -606TT homozygotes. Stratification analyses showed that a reduced risk associated with the -606CC genotype was more pronounced in subgroups of non-smokers, non-drinkers, younger subjects (defined as ≤ 57 years), carriers of TP53 Arg/Arg (rs1042522) genotype, patients with oropharyngeal cancer or late-stage SCCHN. Further in silico analysis revealed that the -249 T-to-C change led to a gain of a transcription factor binding site. Additional functional analysis showed that the -249T-to-C change significantly enhanced transcriptional activity of the DEC1 promoter and the DNA-protein binding activity. We conclude that the DEC1 promoter -249 T>C (rs2012775) polymorphism is functional, modulating susceptibility to SCCHN among non-Hispanic Whites. Additional large-scale, preferably population-based studies are needed to validate our findings.^

CHEMOSENSITIZATION OF HEPATOCELLULAR CARCINOMA TO GEMCITABINE BY NON-INVASIVE RADIOFREQUENCY FIELD-INDUCED HYPERTHERMIA

Gemcitabine is a potent nucleoside analogue against solid tumors however drug resistance rapidly emerges. Removal of gemcitabine incorporated in the DNA by repair mechanisms could potentially contribute to resistance in chemo-refractory solid tumors. In this study, we evaluated homologous recombination repair of gemcitabine-stalled replication forks as a potential mechanism contributing to resistance. We also studied the effect of hyperthermia on homologous recombination pathway to explain the previously reported synergy between gemcitabine and hyperthermia. We found that hyperthermia degrades and inhibits localization of Mre11 to gemcitabine-stalled replication forks. Furthermore, gemcitabine-treated cells that were also treated with hyperthermia demonstrate a prolonged passage through late S/ G2 phase of cell cycle in comparison to cells treated with gemcitabine alone. This coincides with inhibition of resolution of γH2AX foci. Our findings also demonstrate that thermal sensitization of human hepatocellular carcinoma cell lines to gemcitabine is mediated through an Mre11-dependent homologous recombination repair pathway. Combination of non-invasive radiofrequency field-induced hyperthermia and gemcitabine was superior to either therapy alone (p

Pathologic Markers of Prognosis in Ampullary Carcinoma

Ampullary cancer is a rare gastrointestinal malignancy that can be curable with surgical resection of localized disease. The benefit of adjuvant therapy, however, remains unknown in these patients partly because of difficulty in stratifying which patients are at high risk for recurrence. To better identify those patients who may benefit from adjuvant therapy, I conducted a retrospective analysis the pathology reports from 176 patients with surgically resected ampullary cancer who had not received any neoadjuvant therapy, the systemic therapy given, and the patient outcomes. A tissue microarray (TMA) of 95 surgically resected ampullary specimens was also constructed to examine whether there is a correlation between classical immunohistochemical profiles for intestinal and pancreaticobiliary tumors and their histologic classification. In this study, I confirmed the prognostic value of advanced T-stage, nodal metastases, and lymphovascular invasion. Patients whose tumors had “high risk” features had a significantly worse overall survival (p=.002). Furthermore, my research highlighted the importance of histology and its impact on survival, with pancreaticobiliary-like features being a negative prognostic factor (p=0.001). Importantly, patients whose tumors have pancreaticobiliary histology appear to benefit from adjuvant therapy, further implicating histology as an important pathologic marker (p=0.053). In addition, the TMA confirmed a correlation between classical immunohistochemical profiles for intestinal and pancreaticobiliary tumors and histologic classification. My research findings suggest that histology subtypes, T-stage, nodal metastases, and lymphovascular invasion should all be taken into consideration when determining which patients with ampullary cancer may benefit from further adjuvant therapy.

Platelets and Anti-Angiogenic Resistance in Ovarian Carcinoma

Background: Resistance to targeted anti-angiogenic therapy is a growing clinical concern given the disappointing clinical impact of anti-angiogenic. Platelets represent a component of the tumor microenvironment that are implicated in metastasis and represent a significant reservoir of angiogenic regulators. Thrombocytosis has been shown to be caused by malignancy and associated with adverse clinical outcomes, however the causal connections between these associations remain to be identified. Materials and Methods: Following IRB approval, patient data were collected on patients from four U.S. centers and platelet levels through and after therapy were considered as indicators of recurrence of disease. In vitro effects of platelets on cancer cell proliferation, apoptosis, and migration were examined. RNA interference was used to query signaling pathways mediating these effects. The necessity of platelet activation for in vitro effect was analyzed. In vivo orthotopic models were used to query the impact of thrombocytosis and thrombocytopenia on the efficacy of cytotoxic chemotherapy, the effect of aspirin on thrombocytosis and cancer, and platelet effect on anti-angiogenic therapy. Results: Platelets were found to increase at the time of diagnosis of ovarian cancer recurrence in a pattern comparable to CA-125. Platelet co-culture increased proliferation, increased migration, and decreased apoptosis in all cell lines tested. RNA interference implicated platelet derived growth factor alpha (PDGFRA) and transforming growth factor beta-receptor 1 (TGFBR1) signaling. Biodistribution studies suggested minimal platelet sequestration of taxanes. Blockade of platelet activation blocked in vitro effects. In vivo, thrombocytosis blocked chemotherapeutic efficacy, thrombocytopenia increased chemotherapeutic efficacy, and aspirin therapy partially blocked the effects of thrombocytosis. In vivo, withdrawal of anti-angiogenic therapy caused loss of therapeutic benefit with evidence of accelerated disease growth. This effect was blocked by use of a small-molecule inhibitor of Focal Adhesion Kinase. Anti-angiogenic therapy was also associated with increased platelet infiltration into tumor that was not seen to the same degree in the control or FAK-inhibitor-treated mice. Conclusions: Platelets are active participants in the growth and metastasis of tumor, both directly and via facilitation of angiogenesis. Blocking platelets, blocking platelet activation, and blocking platelet trafficking into tumor are novel therapeutic avenues supported by this data. Copyright © 2012 Justin Neal Bottsford-Miller, all rights reserved.

Association of p21/p27 polymorphisms with risk of HPV16-associated oral squamous cell carcinoma

The increasing incidence of oral squamous cell carcinoma (OSCC) among young adults has been associated with sexually transmitted infection of human papillomavirus (HPV), particularly HPV16. Given the roles of p21 (WAF1/Cip1/CDKN1A) and p27 (Kip1/CDKNIB) in cell-cycle regulation and of HPV16 E6 and E7 oncoproteins in p53 degradation and pRb inactivation, the effect of HPV16 L1 seropositivity and three putatively functional single-nucleotide polymorphisms (SNPs) of p21 (p21 C70T and p21 C98A) and p27 (p27 T109G), individually and in combination, on the risk of OSCC was evaluated in a hospital-based case-control study of 327 cases and 401 cancer-free controls who were frequency-matched on age, gender and smoking status. Individuals with HPV16 L1 seropositivity had an overall 3-fold increased risk of having OSCC than those with HPV16 seronegativity. The increased risk of HPV16-associated OSCC was particularly found among younger people (aged ≤ 50 years), males, never smokers, never drinkers and oropharynx cancer patients. None of three p21 and p27 polymorphisms alone was significantly associated with risk of OSCC. Individuals with variant genotypes for both p21 polymorphisms were more likely to have OSCC than individuals with wild-type genotypes or variant genotypes for either one of the p21 polymorphisms (adjusted OR, 1.4; 95% CI, 0.9-2.1). There was a borderline significant or significant interaction between the p21 C70T, combined p21 and combined p21/p27 genotypes and HPV16 L1 seropositivity on risk of OSCC. The three studied p21 and p27 polymorphisms, individually or in combination, did not appear to have an effect on HPV16-related clinical outcomes (overall and disease-free survival and tumor recurrence). Despite the fact that the exact biological mechanism remains to be explored, these findings suggest possible involvement of p21variants, particularly the p21 C70T variant genotypes (CT/TT), in the etiology of HPV16-associated OPSCC. Further large and functional studies are required to validate the findings.^

Role of obesity, weight gain, physical activity, and polymorphisms in the mTOR pathway and the risk of renal cell carcinoma

The interplay between obesity, physical activity, weight gain and genetic variants in mTOR pathway have not been studied in renal cell carcinoma (RCC). We examined the associations between obesity, weight gain, physical activity and RCC risk. We also analyzed whether genetic variants in the mTOR pathway could modify the association. Incident renal cell carcinoma cases and healthy controls were recruited from the University of Texas MD Anderson Cancer Center in Houston, Texas. Cases and controls were frequency-matched by age (±5 years), ethnicity, sex, and county of residence. Epidemiologic data were collected via in-person interview. A total of 577 cases and 593 healthy controls (all white) were included. One hundred ninety-two (192) SNPs from 22 genes were available and their genotyping data were extracted from previous genome-wide association studies. Logistic regression and regression spline were performed to obtain odds ratios. Obesity at age 20, 40, and 3 years prior to diagnosis/recruitment, and moderate and large weight gain from age 20 to 40 were each significantly associated with increased RCC risk. Low physical activity was associated with a 4.08-fold (95% CI: 2.92-5.70) increased risk. Five single nucleotide polymorphisms (SNPs) were significantly associated with RCC risk and their cumulative effect increased the risk by up to 72% (95% CI: 1.20-2.46). Strata specific effects for weight change and genotyping cumulative groups were observed. However, no interaction was suggested by our study. In conclusion, energy balance related risk factors and genetic variants in the mTOR pathway may jointly influence susceptibility to RCC. ^

Factors associated with HPV16 status and survival among patients with squamous cell carcinoma of the oropharynx

Background: Squamous cell carcinoma of the oropharynx (SCCOP) is characterized by local tumor aggressiveness, high recurrence rates, a high incidence of second primary tumors, and medical comorbidities. Significant trends in demographic and clinical characteristics as well as survival among SCCOP patients have been observed over time, likely owing to the changing etiology of the disease. Human papillomavirus type 16 (HPV16) infection is associated with a significant proportion of these cancers. Biomarkers that may aid in identifying patients that are at higher risk of recurrence and death are important so that these patients may be followed more closely to improve their quality of life. ^ Study population and methods: The retrospective review (Specific Aim 2) included 3891 newly diagnosed, previously untreated patients presenting to our institution between 1955 and 2004. A total of 2299 patients treated at our institution were included in survival and recursive partitioning analysis. The prospective cohort study (Specific Aim 3) included 266 patients presenting to our institution between January 2006 and September 2009. ^ Results: The results from the retrospective review showed that over time, patients presented at younger ages and were more likely to have base of tongue/tonsil tumors and to be never/former smokers and moreover survival improved significantly over time. In survival and recursive partitioning analyses, the TNM staging system was efficient in prognosticating patients prior to 1995. However, in the recent decade, the TNM staging system was completely inadequate. The factors having the greatest positive effect on overall survival since 1995 were those common to HPV-associated SCCOP. The results from the prospective cohort study indicate that patients with high nodal stage and those with late stage disease have increased levels of pretreatment serum HPV DNA. ^ Conclusions: We saw a distinct improvement in survival among SCCOP patients over the past 50 years at our institution. The main factors contributing to this were changes in clinical characteristics, in particular surrogates for HPV status. The current TNM staging system for SCCOP is inadequate and incorporation of HPV status (and perhaps smoking status) is encouraged. Furthermore, although pretreatment circulating levels of HPV DNA was associated with higher N category and overall disease stage, it has limited utility as a marker for recurrence among SCCOP patients.^

Characterization of Jak, STAT, and Src interactions in Head and Neck Squamous Cell Carcinoma

Recurrence of Head and Neck Squamous Cell Carcinoma (HNSCC) is common; thus, it is essential to improve the effectiveness and reduce toxicity of current treatments. Proteins in the Src/Jak/STAT pathway represent potential therapeutic targets, as this pathway is hyperactive in HNSCC and it has roles in cell migration, metastasis, proliferation, survival, and angiogenesis. During short-term Src inhibition, Janus kinase (Jak) 2, and signal transducer and activator of transcription (STAT) 3 and STAT5 are dephosphorylated and inactivated. Following sustained Src inhibition, STAT5 remains inactive, but Jak2 and STAT3 are reactivated following their early inhibition. To further characterize the mechanism of this novel feedback pathway we performed several experiments to look at the interactions between Src, Jak2, STAT5 and STAT3. We attempted to develop a non-radioactive kinase assay using purified recombinant Jak2 and Src proteins, but found that phospho-tyrosine antibodies were non-specifically binding to purified recombinant proteins. We then performed in vitro kinase assays (IVKAs) using purified recombinant Jak2, Src, STAT3, and STAT5 proteins with and without Src and Jak2 pharmacologic inhibitors. We also examined the interactions of these proteins in intact HNSCC cells. We found that recombinant Jak2, STAT3, and STAT5 are direct substrates of Src and that recombinant Src, STAT3, and STAT5 are direct substrates of Jak2 in the IVKA. To our knowledge, the finding that Src is a Jak substrate is novel and has not been shown before. In intact HNSCC cells we find that STAT3 can be reactivated despite continuous Src inhibition and that STAT5 continues to be inhibited despite Jak2 reactivation. Also, Jak2 inhibition did not affect Src or STAT5 activity but it did cause STAT3 inhibition. We hypothesized that the differences between the intact cells and the IVKA assays were due to a potential need for binding partners in intact HNSCC cells. One potential binding partner that we examined is the epidermal growth factor receptor (EGFR). We found that EGFR activation caused increased activation of Src and STAT5 but not Jak2. Our results demonstrate that although STAT3 and STAT5 are capable of being Src and Jak2 substrates, in intact HNSCC cells Src predominantly regulates STAT5 and Jak2 regulates STAT3. Regulation of STAT5 by Src may involve interactions between Src and EGFR. This knowledge along with future studies will better define the mechanisms of STAT regulation in HNSCC cells and ultimately result in an ideal combination of therapeutic agents for HNSCC.

The risk prediction for SPM and its impact on the survival in patients with head and neck squamous cell carcinoma

Head and Neck Squamous Cell Carcinoma (HNSCC) is the sixth common malignancy in the world, with high rates of developing second primary malignancy (SPM) and moderately low survival rates. This disease has become an enormous challenge in the cancer research and treatments. For HNSCC patients, a highly significant cause of post-treatment mortality and morbidity is the development of SPM. Hence, assessment of predicting the risk for the development of SPM would be very helpful for patients, clinicians and policy makers to estimate the survival of patients with HNSCC. In this study, we built a prognostic model to predict the risk of developing SPM in patients with newly diagnosed HNSCC. The dataset used in this research was obtained from The University of Texas MD Anderson Cancer Center. For the first aim, we used stepwise logistic regression to identify the prognostic factors for the development of SPM. Our final model contained cancer site and overall cancer stage as our risk factors for SPM. The Hosmer-Lemeshow test (p-value= 0.15>0.05) showed the final prognostic model fit the data well. The area under the ROC curve was 0.72 that suggested the discrimination ability of our model was acceptable. The internal validation confirmed the prognostic model was a good fit and the final prognostic model would not over optimistically predict the risk of SPM. This model needs external validation by using large data sample size before it can be generalized to predict SPM risk for other HNSCC patients. For the second aim, we utilized a multistate survival analysis approach to estimate the probability of death for HNSCC patients taking into consideration of the possibility of SPM. Patients without SPM were associated with longer survival. These findings suggest that the development of SPM could be a predictor of survival rates among the patients with HNSCC.^

Mechanisms of multidrug resistance: Differences between natural and acquired adriamycin -resistant human colon carcinoma cells

Mechanisms of multidrug resistance (MDR) were studied in two independent MDR sublines (AdR1.2 and SRA1.2) derived from the established human colon carcinoma cell line LoVo. AdR1.2 was developed by long-term continuous exposure of the cells to adriamycin (AdR) in stepwise increments of concentration, while SRA1.2 was selected by repetitive pulse treatments with AdR at a single concentration. In this dissertation, the hypothesis that the mechanism of drug resistance in SRA1.2 is different than that in AdR1.2 is tested. While SRA1.2 demonstrated similar biological characteristics when compared to the parental LoVo, AdR1.2 exhibited remarkable alterations in biological properties. The resistance phenotype of AdR1.2 was reversible when the cells were grown in the drug-free medium whereas SRA1.2 maintained its resistance for at least 10 months under similar conditions. Km and Vmax of carrier-mediated facilitated diffusion AdR transport were similar among the three lines. However, both resistant sublines exhibited an energy-dependent drug efflux. AdR1.2 appeared to possess an activated efflux pump, and a decreased nucleus-binding of AdR, whereas SRA1.2 showed merely a lower affinity in binding of AdR to the nuclei. Southern blot analysis showed no amplification of the MDR1 gene in either of the two resistant subclones. However, Western blot analysis using the C219 monoclonal antibody against P170 glycoprotein detected a Mr 150-kDa plasma protein (P150) in AdR1.2 but not in SRA1.2 or in the parental LoVo. In vitro phosphorylation studies revealed that P150 was a phosphoprotein; its phosphorylation was Mg$\sp{2+}$-dependent and could be enhanced by verapamil, an agent capable of increasing intracellular AdR accumulation in AdR1.2 cells. The phosphorylation studies also revealed elevated phosphorylation of a Mr 66-kDa plasma membrane protein that was detectable in the AdR1.2 revertant and in AdR1.2 when verapamil was present, suggesting that hyperphosphorylation of the Mr 66-kDa protein may be related to the reversal of MDR. SDS-PAGE of the plasma membrane protein demonstrated overproduction of a Mr 130-kDa, MDR-related protein in both the resistant sublines. The Mr 130-kDa, MDR-related protein in both the resistant sublines. The Mr 130-kDa protein was not immunoreactive with C219, but its absence in the AdR1.2 revertant and the parental LoVo suggests that it is an MDR-related plasma membrane protein. In conclusion, the results from this study support the author's hypothesis that the mechanisms responsible for "Acquired" and "Natural" MDR are not identical. ^

Modulation of carbohydrates and carbohydrate binding proteins (lectins) during retinoic acid -induced differentiation of murine embryonal carcinoma cells

The current studies were undertaken to examine the effect of retinoic acid (RA)-induced differentiation of the murine embryonal carcinoma cell line, F-9, on the glycosylation of specific cellular glycoproteins and on the expression of two members of the family of endogenous lactoside-binding lectins. It was found that RA-induced differentiation of these cells into cells with the properties of primitive endoderm results in the increased fucosylation of 3 glycoproteins with molecular weights of 175 (gp175), 250 (gp250), and 400 (pg400) kDa. These three fucose-containing glycoproteins can be considered as new markers of differentiation in this system. The increased fucosylation of these glycoproteins preceded the 3-fold increase in fucosyltransferase (FT) activity that was seen upon RA-induced differentiation of these cells, indicating that an increase in fucosyltransferase activity alone cannot explain the increased fucosylation of these glycoproteins.^ The effect of RA and Ch55, a chalcone carboxylic acid with retinoid-like properties, induced differentiation of a variety of murine embryonal carcinoma cell lines on the activities of both FT and sialyltransferase (ST) was examined. The effect of differentiation on the activities of both glycosyltransferases was modulated and most probably is dependent upon the differentiation pathway that is triggered by the retinoids for each of the embryonal carcinoma cell lines.^ Two glycoproteins, Lysosomal Associated Membrane Glycoproteins 1 and 2 (LAMP-1 and LAMP-2) were examined in more detail during the course of RA-induced differentiation of F-9 cells. Both the levels and glycosylation of both glycoproteins are increased following differentiation of these cells. Differentiation results in the increased binding of $\sp{125}$l-labelled L-phytohemagglutinin to bind to LAMP-1 which indicates increased GlcNAc $\beta$1,6 branching of the oligosaccharide side chains.^ We found that RA-induced differentiation of F-9 cells results in the decreased expression of the 34 kDa lectin 24 h after addition of the retinoid to the medium. Additionally, 48 h of RA-treatment results in the increased expression of the 14.5 kDa lectin. By indirect immunofluorescence we were able to colocalize the 14.5 kDa lectin and laminin which suggests that laminin may be a ligand for the lectin in the F-9 cells. (Abstract shortened with permission of author.) ^