Research Interests
My research program centers on exploring the cellular and molecular changes initiated by human papillomavirus type 16 (HPV16), a DNA virus associated with nearly all cervical cancers. In particular we use an in vitro model system of HPV-mediated human cell carcinogenesis. Using this model we have characterized several key changes that mark the progression from normal to premalignant cells upon transfection of normal human keratinocytes (HKc) with HPV16 DNA. We have determined that progression in this model is associated with increasing resistance to growth inhibition by transforming growth factor-beta (TGF-beta)?. TGF-beta resistance is the result of a loss in the expression of the TGF-beta receptor type I (TGF-beta RI). Current studies, funded by the National Cancer Institute (NCI), are exploring the molecular mechanism(s) leading to a loss of TGF-beta RI in these cells.
Additional areas of focus in my laboratory include; (1) studies exploring the specific functions of the HPV16 E6 and E7 oncoproteins required to induce TGF-beta resistance and disrupt TGF-beta signaling in normal HKc and (2) experiments aimed at identifying the elements within the HPV16 upstream regulatory region (URR) that are responsible for TGF-beta modulation of HPV16 early gene expression. Our recent results, published in the Journal of Virology, demonstrate that TGF-beta modulation of HPV16 early gene expression is mediated by NF1/Ski interactions.
A long term goal of our research is to determine which markers of progression identified in our in vitro model can be verified and tested in the human population. To this end we are employing a DNA microarray approach, using both our in vitro model system and cervical cells collected from routine Pap smears. These studies are being conducted in our microarray core facility, which I serve as Director. We hope to identify biological markers of cervical cancer progression that will find an application in screening, using “diagnostic” DNA microarrays, to ultimately identify among HPV positive women, those at greatest risk to develop cervical cancer.
Finally, in a program recently funded by the NCI, I am coordinating a project that brings together Claflin University (a Historically Black College or University located in Orangeburg, South Carolina) and the South Carolina Cancer Center in a collaborative effort aimed at training minority undergraduate students in cancer research, and at establishing solid collaborative research programs among cancer researchers at the two institutions.
Recent Publications
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- Rapid Commun Mass Spectrom. 2001;15(18):1752-9
Genotyping single nucleotide polymorphisms using intact polymerase chain reaction products by electrospray quadrupole mass spectrometry.
Walters JJ, Muhammad W, Fox KF, Fox A, Xie D, Creek KE, Pirisi L.
Both single nucleotide polymorphisms (SNPs) and mutations are commonly observed in the gene encoding the tumor suppressor protein, p53. SNPs occur at specific locations within genes whereas mutations may be distributed across large regions of genes. When determining nucleotide differences, mass spectrometry is the only method other than Sanger sequencing which offers direct structural information. Electrospray ionization (ESI) quadrupole mass spectrometry (MS) analysis of intact polymerase chain reaction (PCR) products was performed following a simple purification and on-line heating to limit ion adduction. The PCR products were amplified directly from genomic DNA rather than plasmids, as in our previous work. Two known polymorphisms of the p53 gene were genotyped. A cytosine (C) or guanine (G) transversion, designated C <--> G (G <--> C on the opposite strand), were each detected by a 40.0 Da change upon ESI quadrupole MS analysis. Using known PCR products as standards, the genotypes determined for 10 human samples corresponded with restriction fragment length polymorphism (RFLP) analysis. Cytosine/thymine (T) transitions, designated C <--> T (G <--> A on the opposite strand), were also genotyped by ESI-MS. This SNP is discriminated by a 15.0 Da change on one strand (C <--> T) and a 16.0 Da change on the other (G <--> A). Appropriate sample preparation and instrumental configuration (including heated sample inlet syringe and MS source), to limit adducts, are both vital for successful ESI quadrupole MS analysis of intact PCR products. Copyright 2001 John Wiley & Sons, Ltd.
- Cancer Res. 2001 May 1;61(9):3837-43.
Human papillomavirus type 16 E6 and E7 cooperate to increase epidermal growth factor receptor (EGFR) mRNA levels, overcoming mechanisms by which excessive EGFR signaling shortens the life span of normal human keratinocytes.
Akerman GS, Tolleson WH, Brown KL, Zyzak LL, Mourateva E, Engin TS, Basaraba A, Coker AL, Creek KE, Pirisi L.
Epidermal growth factor receptor (EGFR) levels are dramatically increased in human keratinocytes (HKc) immortalized with full-length human papillomavirus type 16 (HPV16) DNA (HKc/HPV16), but increases in EGFR levels actually precede immortalization. In some normal HKc strains, acute expression of HPV16 E6 (but not HPV16 E5, HPV16 E7, or HPV6 E6) from LXSN retroviral vectors produced an increase in EGFR mRNA levels detectable at 24 h and stable for up to 10 days after infection. However, about one-half of the individual normal HKc strains we analyzed proved unresponsive to E6 induction of EGFR mRNA despite the robust expression of E6 and degradation of p53. E6 responsiveness of normal HKc strains correlated inversely with initial EGFR levels: although HKc strains expressing relatively low basal EGFR levels grew poorly and tolerated the infection protocol with difficulty, they responded to E6 with an increase in EGFR mRNA and protein and with robust proliferation. However, those HKc strains expressing high basal EGFR levels grew well, but did not respond to E6 with increased EGFR levels or with proliferation. Immunostaining of paraffin-embedded foreskin tissue for the EGFR confirmed that there is an intrinsic interindividual variability of EGFR expression in HKC: These results prompted us to investigate the effects of overexpression of the EGFR in normal HKC: Infection of normal HKc with a LXSN retrovirus expressing the full-length human EGFR cDNA resulted in a dramatic reduction in growth rate and a shorter life span. Although acute expression (1-10 days after infection) of HPV16 E7 alone did not induce the EGFR, acute expression of E6 and E7 together increased EGFR levels in normal HKc unresponsive to E6 alone. Also, HKc infected with E7 alone expressed increased EGFR levels at early stages of extended life span (at passage 9 after infection), and HKc immortalized by HPV16 E7 alone expressed EGFR levels comparable with those of E6/E7-immortalized cells. These results support a key role of the EGFR in HPV16-mediated transformation of HKC: In addition, these data show that normal HKc do not tolerate excessive EGFR levels/signaling, and such intolerance must be overcome in order for HKc to become immortalized by HPV16. We conclude that both E6 and E7 contribute to increasing EGFR levels, but with different mechanisms: although E6 can increase EGFR levels, it cannot overcome the resistance of normal HKc to excessive EGFR signaling. On the other hand E7, which alone does not acutely increase EGFR mRNA or protein, allows for EGFR overexpression in normal HKC
- Virology 2000 May 10;270(2):408-16
Loss of transforming growth factor-beta (TGF-beta) receptor type I mediates TGF-beta resistance in human papillomavirus type 16-transformed human keratinocytes at late stages of in vitro progression.
Mi Y, Borger DR, Fernandes PR, Pirisi L, Creek KE.
Human keratinocytes (HKc) immortalized by human papillomavirus type 16 DNA (HKc/HPV16) progress toward malignancy through growth factor-independent (HKc/GFI) and differentiation-resistant stages (HKc/DR). This progression is associated with a loss of sensitivity to growth inhibition by both all-trans-retinoic acid (RA) and transforming growth factor-beta (TGF-beta). In the accompanying article (Borger et al., 2000, Virology 270, 397-407), we demonstrate that RA resistance in HKc/HPV16 arises despite functional nuclear retinoid receptors and that TGF-beta mediates growth inhibition by RA. To investigate the basis for the loss of TGF-beta sensitivity during in vitro progression of HKc/HPV16, we explored the expression of TGF-beta receptors type I and type II in independently derived HKc/HPV16 lines and their corresponding HKc/GFI and HKc/DR derivatives. While TGF-beta receptor type II mRNA levels were unchanged during progression, mRNA levels for TGF-beta receptor type I decreased dramatically as the cells became TGF-beta resistant. At the HKc/DR stage, loss of TGF-beta receptor type I mRNA, compared to low-passage cells, ranged from 55 to 87% in four HKc/HPV16 lines examined. Immunohistochemistry, using anti-TGF-beta receptor type I antibodies, confirmed a loss of TGF-beta receptor type I expression in HKc/DR. Reintroduction of the TGF-beta-receptor type I into TGF-beta-resistant HKc/DR completely restored growth inhibition by TGF-beta. Southern blot analysis of DNA extracted from normal HKc, HKc/HPV16, and HKc/DR ruled out any gross changes in the TGF-beta receptor type I gene. The activity of the TGF-beta receptor type I promoter, cloned upstream of a luciferase reporter gene, was decreased in HKc/DR, to an extent comparable to the decrease in mRNA levels for the TGF-beta receptor type I. Thus, TGF-beta resistance at late stages of HPV16-mediated transformation of HKc is the result of a loss of expression of TGF-beta receptor type I.
- Oncogene 2000 Jun 29;19(28):3172-81
Unique carboxyl-terminal sequences of wild type and alternatively spliced variant forms of transforming growth factor-alpha precursors mediate specific interactions with ErbB4 and ErbB2. Xu X, Kelleher KF, Liao J, Creek KE, Pirisi L.
We have previously reported that the human transforming growth factor-alpha (TGF-alpha) gene encodes three forms of TGF-alpha precursors, designated wild type (WT), variant I (VaI), and variant II (VaII), derived from alternative splicing. The two carboxyl-terminal valine residues of WT are replaced by 5 (GCRLY) or 4 (ATLG) amino acids in VaI or VaII, respectively. When overexpressed in Chinese hamster ovary (CHO) cells, VaI and ValI, but not WT, support autonomous growth. We detected tyrosine phosphorylation of ErbB2 in the absence of serum, in CHO cells expressing WT, VaI, or VaII, but not in mock transfectants. These observations prompted us to investigate possible interactions between the ErbBs and the TGF-alpha precursors in CHO cells. All TGF-alpha precursors were found to co-immunoprecipitate with the ErbBs, but with different specificity. WT co-immunoprecipitated with ErbB4, but not with ErbB1, ErbB2, or ErbB3. VaI and VaII co-immunoprecipitated with ErbB2, but not with ErbB1, ErbB3, or ErbB4. Confocal fluorescent microscopy analysis demonstrated that WT, VaI, and VaII all distribute equally to the cell surface while, as expected, a WT mutant lacking the two C-terminal valine residues does not. Point and deletion mutants involving the unique carboxyl-terminal residues of WT, VaI and VaII, indicated that the interactions between the three TGF-alpha precursors and the ErbBs were mediated by their carboxyl-terminal regions, which constitute distinct protein-binding motifs. A chimera of the intracellular domain of WT TGF-alpha linked to exogenous transmembrane and extracellular domains retained both the cell surface distribution and the specific interaction with ErbB4 of full-length WT, confirming that this interaction is mediated by the C-terminus of the TGF-alpha precursor. While interactions of WT and variant TGF-alpha with the ErbBs all result in ErbB2 activation, they produce different biological consequences, suggesting that the various TGF-alpha precursors differentially modulate ErbB signaling.
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