Search PubMed for publications by Dr Kim Creek
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. |
