ORIGINAL PAPER

Physical Labeling of Papillomavirus-Infected, Immortal,and Cancerous Cervical Epithelial Cells Reveal Surface Changesat Immortal Stage

K. Swaminathan Iyer • R. M. Gaikwad •

C. D. Woodworth • D. O. Volkov • Igor Sokolov

Published online: 17 February 2012

� Springer Science+Business Media, LLC 2012

Abstract A significant change of surface features of

malignant cervical epithelial cells compared to normal

cells has been previously reported. Here, we are studying

the question at which progressive stage leading to cervical

cancer the surface alteration happens. A non-traditional

method to identify malignant cervical epithelial cells in

vitro, which is based on physical (in contrast to specific

biochemical) labelling of cells with fluorescent silica

micron-size beads, is used here to examine cells at pro-

gressive stages leading to cervical cancer which include

normal epithelial cells, cells infected with human papillo-

mavirus type-16 (HPV-16), cells immortalized by HPV-16,

and carcinoma cells. The study shows a statistically sig-

nificant (at p \ 0.01) difference between both immortal

and cancer cells and a group consisting of normal and

infected. There is no significant difference between normal

and infected cells. Immortal cells demonstrate the signal

which is closer to cancer cells than to either normal or

infected cells. This implies that the cell surface, surface

cellular brush changes substantially when cells become

immortal. Physical labeling of the cell surface represents a

substantial departure from the traditional biochemical

labeling methods. The results presented show the potential

significance of physical properties of the cell surface for

development of clinical methods for early detection of

cervical cancer, even at the stage of immortalized, pre-

malignant cells.

Keywords Cervical cancer � Early cancer detection �Novel detection methods � Fluorescent silica particles

Introduction

Cervical cancer is the second most common cause of

cancer death in women worldwide; infection with onco-

genic human papillomavirus (HPV) is the most significant

risk factor in its etiology [1]. The mortality rate is second

only to that for breast cancer. Early detection of this cancer

can substantially decrease mortality due to this disease.

The demand to reduce the mortality related to cervical

cancer has resulted in inter-disciplinary research involving

physics, chemistry, molecular biology, engineering, and

medicine [2]. Research efforts over the years have been

directed toward the development of a universal, reliable,

and consistent test to detect malignant cells at the early

stages of cancer development, and in particular, prema-

lignant cervical cells, also known as cervical intraepithelial

neoplasia (CIN) [3].

Electronic supplementary material The online version of thisarticle (doi:10.1007/s12013-012-9345-2) contains supplementarymaterial, which is available to authorized users.

K. Swaminathan Iyer � R. M. Gaikwad �D. O. Volkov � I. Sokolov (&)

Department of Physics, Clarkson University, Potsdam,

NY 13699-5820, USA

e-mail: isokolov@clarkson.edu

Present Address:K. Swaminathan Iyer

School of Biomedical, Biomolecular and Chemical Sciences,

The University of Western Australia, Crawley, WA, Australia

C. D. Woodworth

Department of Biology, Nanoengineering and Biotechnology

Laboratories Center (NABLAB), Clarkson University,

Potsdam, NY 13699-5820, USA

I. Sokolov

Nanoengineering and Biotechnology Laboratories Center

(NABLAB), Clarkson University, Potsdam,

NY 13699-5820, USA

123

Cell Biochem Biophys (2012) 63:109–116

DOI 10.1007/s12013-012-9345-2

HPV-associated cervical carcinogenesis is a multistep

process leading to the transformation of the infected cells

to CIN [3]. The cells associated with the lesions become

atypical and mitotically active, and they are characterized

by the inability to terminally differentiate [4]. Low grade

CIN are typically limited to the lower portion of the epi-

thelia, but they can progress to high-grade CIN and become

evident throughout the entire thickness of the epithelia and

eventually lead to cervical carcinoma. The multistep pro-

cess leading to transformation of infected cells to CIN is

associated with over expression of two early genes, E6 and

E7 [5, 6]. These genes are consistently expressed in cer-

vical cancer cell lines and involved in HPV-mediated

carcinogenesis [7]. The transforming ability of the genes is

partly due to their ability to interact with cellular tumor

suppressor proteins. E6 binds the tumor suppressor p53

through its interaction with another cellular protein, E6-

associated protein (E6-AP), leads to degradation of p53 via

the ubiquitin-mediated protein degradation pathway [8, 9].

E7 binds and degrades the retinoblastoma tumor suscepti-

bility gene product (pRb) and also the other pRb ‘‘pocket’’

protein family members, p107 and p130 [10, 11].

The Papanicolaou (Pap) smear, a cytological screening

test has been one of the most successful methods of cer-

vical cancer detection over the years [12]. Although the

Pap smear is the most widely used cancer screening

method in the world and its impact on the incidence of

cervical cancer is well known from a historical perspective,

recent reports suggest that the sensitivity of Pap smear

screening is 50–80%, with the relative proportion of sam-

pling to screening errors being 2:1 [13]. There are multiple

benign mimics of neoplastic cells such as atypia of repair,

atrophy, radiation change, effect of intrauterine device, and

metaplasia. These cells are called atypical cells of unde-

termined significance due to the inability of optical

microscopy to identify definite features of either benign or

neoplastic cells. The tests may be further complicated by

high-unsatisfactory rates, preparation artifacts, and unnec-

essary cost interventions. Each year in the United States

alone approximately 3.5 million Pap smears cytological

tests are classified as equivocal, out of which only 8% of

women will have preinvasive (high-grade squamous

intraepithelial) lesions, and 0.4% will have carcinoma as

found in further testing that involves invasive tissue biop-

sies. DNA tests (detection of HPV, human papillomavirus

infection) are rather accurate, but identify only a risk of

cancer (the fact of HPV infection) but not cancer [44].

The economic constraints in developing countries have

prompted alternative methods of screening for cancer

including visual inspection after application of 3–5% of

acetic acid [14] and Lugol’s iodine [15]. The major dis-

advantage of these tests is low specificity. Given the con-

siderable variation in the way these tests are applied and

interpreted in different settings, there is no standard uni-

versally accepted definition of the test results. It remains to

be seen if the specificity can be improved by further

developments in test definitions and training strategies. The

inability of these test methods to unambiguously detect

abnormal cells during the early stages, poses a major

challenge in an effort to reduce mortality associated with

cervical cancer.

Demands for the development of a universal testing and

screening method have generated enormous scientific and

industrial interest in the past. Several methods such as

organic receptor molecules that undergo color changes

[16], electrochemical, [17] magnetic [18], and fluorescent

tags [19, 20] have been developed and reported. Optical

techniques like fluorescent microscopy [21] confocal

imaging [22] and optical coherence tomography (OCT)

[23] have shown promise for cervical precancer detection

as measured against the traditional methods such a Pap

smear. The sensitivity and specificities of these optical

techniques have been reported to reach 80–90% in small

and moderate clinical trials [24].

Among the optical techniques, detection using fluores-

cence is an important non-isotopic method. The fluorescent

biological labels have played an important role in the field

of biomedicine, such as the detection of materials inside or

outside of the cell [25, 26], hybridization and sequencing of

nucleic acids [27], and clinical diagnosis at the early stage

[28]. These conventional fluorescent techniques have the

following limitations: reduction in the emission intensity

due to photobleaching, the toxicity of some of the fluo-

rescent dyes may be harmful for living cells, and detection

sensitivity is not so high because only a few fluorophores

can be coupled to one biomolecule in the conventional

fluorescent label methods.

It was recently found [29] that a several micron silica

particle attached to a cantilever of an atomic force micro-

scope [30] (AFM) interacted with cancerous and normal

cells quite differently. The main reason was attributed to a

different topological structure of surface ‘‘brushes’’ cov-

ering cells, which consisted of microvilli, microridges,

cilia, and glycocalyx molecules. Using that observation, a

new method to identify cervical cancer cells was suggested

[31, 32]. The method was based on the use of silica par-

ticles of similar size but fluorescent in nature. The differ-

ence in the cell brush resulted in the different adhesion of

the silica beads-cell. Furthermore, a high-resolution mul-

timodal AFM imaging in novel HarmoniX mode revealed

virtually 100% difference between the surface of normal

and cancerous cells [43]. This AFM method was very

precise but rather slow. In addition, the detection of pre-

malignant (immortal) cells is more interesting for potential

clinical early diagnosis of cancer. Thus, it is important to

test if the faster labeling method Ref. [31] could potentially

110 Cell Biochem Biophys (2012) 63:109–116

123

be effective in the defection of immortal cells. From the

fundamental point of view, it is informative to learn the

stage of cancer progression when the cell surface starts

changing.

In this study, we use the fast method of Ref. [31] to

study cervical cells during the different stages of multistep

transformation leading from normal to cancer. Specifically,

we study normal, infected (with HPV-16), immortal, and

cancerous cells. Here we confirm previous results for

normal and cancerous cervical cells [31], and find that in

the progression leading to cervical cancer, adhesion (or cell

surface) changes significantly at the stage of immortal

cells. We found that cancerous and immortal cells (the

latter were derived from HPV-infected cells) adhere to

silica beads in a statistically different manner than normal

cells and HPV-infected cells.

Materials and Methods

Spectrofluorometric Measurements and Imaging

of Cells

A scheme of the experimental setup is shown in Fig. 1. An

Olympus BH2-UMA microscope, which works in both

transmission and reflection mode, was connected to a JVC

TK-1280U color video camera. The images of cells were

captured using FlashBus MV version 3.91 software. To

record the fluorescent emission, an optic fiber was used to

connect the microscope to an spectrometer (USB 2000 by

Ocean Optics Inc., FL). A Cyonics air-cooled argon ion

laser was used as an excitation source for fluorescence. A

narrow-band 488 nm blocking filter (Omega Optical) was

utilized to filter the laser light. The spectra were recorded

using OOIBase32 software. To avoid excessive focusing on

individual florescent particles, and consequent collecting

the emission spectra from excessively those particles, after

the focusing, the focus knob was rotated three full turns in

counter clockwise direction (defocusing the sample by

increasing the sample-objective lens distance). This pro-

cedure was maintained constant for spectrofluorometric

measurements of all samples. 59 objective was used in

these measurements. This allowed collecting the fluores-

cent signal coming from a predefined area of approxi-

mately 4 mm in diameter (as was found by moving

individual fluorescent beads).

To decrease possible dependency of the fluorescent

signal on the device used, the spectrometer was calibrated

with the help of LS-1 Tungsten Halogen Light Source

(USB 2000 by Ocean Optics Inc., FL) by using the pro-

cedure described by the manufacturer. To decrease the

noise, each fluorescent signal used in this study was an

integral of the fluorescent intensities (taken in the range of

560–680 nm).

Statistical analysis was done with the help of Origin 8

software (Origin Labs). The significance of statistical dif-

ference was analysed with ANOVA test using the confi-

dence level of p of 0.01. Specifically, one-way ANOVA

using the Tukey test of mean comparisons was used. Equal

variances of distributions were verified using Levene’s

tests.

Cell Cultures

Primary cultures of human cervical epithelial cells were

prepared by a two-stage enzymatic digestion of cervical

tissue as described [33] In brief, each tissue was digested

for 16 h at 4�C in dispase and the layer of epithelial cells

was removed from the underlying connective tissue by

scraping. The sheet of epithelial cells was cut into 1 mm2

pieces and digested in 0.25% trypsin at 37�C for 10 min.

Trypsin was neutralized by adding fetal bovine serum and

cells were collected by low-speed centrifugation. Cultures

consisting of C95% epithelial cells were maintained in

keratinocyte serum-free medium (Invitrogen) which pre-

vents outgrowth of fibroblasts and other stromal cells.

HPV-16 immortalized cell lines [41] and cervical carci-

noma cell lines [42] were also maintained in KSFM and no

evidence of contamination by fibroblasts or other stromal

cells was observed.

All human tissue was obtained from the Cooperative

Human Tissue Network. Informed consent was obtained

from patients according to their published guidelines

(http://chtn.nci.nih.gov/phspolicies.html). The transforma-

tion of normal cells in precancerous squamous intraepi-

thelial lesions is associated with over expression of the E6

and E7 genes. HPV genes were introduced into cultured

cervical cells by infection with recombinant retrovirusesFig. 1 (Color online) Schematic diagram of the optical measurement

system

Cell Biochem Biophys (2012) 63:109–116 111

123

encoding HPV-16 E6/E7 genes inserted into the vector

pLXSN, which contains the neomycin resistance gene [34].

Infection (MOI = 10) was performed for 3 h in medium

with 10 ng/ml polybrene with rocking every 15 min.

Subsequently, medium was changed and cells grew for

24 h before cultures were split 1:3. After 24 h, infected

cells were selected by growth for 2 days in KSFM con-

taining 200 lg/ml G418 and used immediately. Approxi-

mately 70–90% of cells were infected as determined by

survival after G418 selection.

Normal cervical cells were used between 40 and 60

population doublings (PDs), and carcinoma cell lines were

used at 90–120. The slightly higher number of PDs for

cancer cell lines avoids potential confusion because any

normal cells (epithelial cells or stromal cells) that may

contaminate the cancer culture dishes would die out by that

number of PDs. This allows us to avoid possible confusion

between cancer cells and either normal epithelial cells or

fibroblasts. All cells were plated in 60 mm tissue culture

dishes and dishes were used for experiments when cells

were 80–100% confluent.

Fluorescent Silica Beads

Recently a new one step self-assembly of nanoporous silica

particles with encapsulated organic dyes has been devel-

oped, [35–37] in which the dyes are physically entrapped

inside silica matrix, inside 2–4 nm in diameter nanochan-

nels. It was found that the synthesized particles could be up

to two orders of magnitude brighter than the micron-size

particles assembled from aqueous dispersible quantum dots

[38] encapsulated in polymeric particles (scaled to the

same size). Comparing with the maximum fluorescence of

free dye in the same volume, the particles can show fluo-

rescence which is higher by a factor of *5,000. This

makes the particles the brightest tags presently available.

The synthesis of these particles is described in the cor-

responding references. In brief, it is a one step synthesis.

Tetraethylorthosilicate (TEOS, 99.99?%, Aldrich), cetyl-

trimethylammonium chloride (CTACl, 25 wt% aqueous

solution, Pflatz & Bauer), formamide (99%, Aldrich) and

hydrochloric acid HCl (37.6 wt% aqueous solution, Safe-

Cote), Rhodamine 640 (R40) dye (Sigma-Aldrige, Inc.)

were used. All chemicals were used as received. The sur-

factant, acid, dye, formamide, and distilled water (Corning,

AG-1b, 1 MX-cm) were stirred in a polypropylene bottle at

room temperature for 2 h, after which TEOS was added

and the solution stirred for ca. 5 min. The solution was then

kept under quiescent conditions for 3 days. The molar ratio

of H2O:HCl:formamide:CTACl:R6G:TEOS was 100:7.8:

9.5:0.11:0.01:0.13. The materials that formed were washed

by centrifugation to avoid damaging of the surface, washed

with copious amounts of water (till the leakage of the dye

stops), and mixed with HBSS solution for the further use

on the cell culture.

Detection of Affinity of Fluorescent Silica Beads

to Cells

After carefully washing the particles to remove any traces

of the synthesizing chemistry, the particles were added to

Hanks balanced salt solution (HBSS) at a pH of 7.4 to form

a colloidal dispersion (*2 g/l). The cells in the 60 mm

culture dishes were washed twice with HBSS solution for

2 min each. The cells were then exposed to 1 ml of the

colloidal dispersion for 2 min. During this 2 min period,

the dishes were subjected to initial 30 s of shaking on a

Boekel Scientific Ocelot 260300F at minimum speed, to

insure uniform distribution of the colloidal solution and to

minimize particle agglomeration. To remove excess parti-

cles that had not adhered to the cells, the cells were then

washed with HBSS solution two times thoroughly using the

same Boekel shaker. The culture dishes were then dried

under ambient conditions at room temperature.

It is important to note the density of cells in the culture

dish. The measurements were done on cells that just reached

confluency in the cell culture dish bottom. This allows us to

exclude the possible affinity of the particles to the culture

dish bottom as well as dealing with different total areas of

coverage of cells in different dishes. In this case, the pro-

cessing of the results becomes simple because the amount

of adherent fluorescent particles is linearly proportional to

the fluorescent signal. The analysis of cells that are far from

confluency is possible [30] although this requires more

processing time or development of a special software.

Results

Figure 2 shows representative optical images (combined

fluorescence and transmission microscopy) of particles that

adhered to the surface of normal, HPV-16 infected,

immortal, and cancerous cells. One can see higher amounts

of particles adherent to cancer and immortal cell surfaces

as compared to normal and infected cells. To describe this

difference quantitatively, we measured fluorescence for

each type of cell. On average, the fluorescent signal

(measured as described in the Materials and Methods

section) is proportional to the number of fluorescent silica

beads adherent to the cell surfaces. Therefore, the fluo-

rescent signal is a good measure of the difference in

adhesion of fluorescent silica beads to the cells.

Figure 3 shows the fluorescent signals of silica beads

measured on culture dishes containing either normal, or

HPV-16 infected, or immortal, or cancerous cells. Indi-

vidual fluorescent spectra and their integral used to obtain

112 Cell Biochem Biophys (2012) 63:109–116

123

the data presented in Fig. 3 are shown in the Supplemen-

tary Materials (Figure S1 and Table S1). Each cell type was

derived from tissues of three human subjects, and was

tested using three culture dishes, 10–20 readings per dish

(5–6 fluorescent spectra averaged over three measurements

each). Figure 3a shows the total statistical data of fluo-

rescence for all human subjects. The average and one

standard deviation are shown. Figure 3b presents the sta-

tistical fluorescence data for each human subject sepa-

rately. The average, one standard deviation and minimum/

maximum are shown (details of the data are provided in the

Supplementary Materials).

One can see an insignificant difference in the fluores-

cence signals from the silica beads adherent on normal and

infected cells. Statistical analysis shows that the difference

in the values of these intensities is statistically insignificant

at the confidence level of 0.01.

Comparing the fluorescence data of the immortal cells

with that of the normal and infected cells, one can see a

difference. It is evident that the fluorescent intensity is

stronger in the case of immortal cells, and getting closer to

that of cancerous cells. Statistical analysis shows that the

fluorescent signal collected from immortal cells is signifi-

cantly different from the signals collected on either normal

or infected cells at the confidence level p \ 0.01.

The same statistically significant difference at the con-

fidence level p \ 0.01 was found for the cancerous cells

compared to the normal, infected cells, and even immortal

cells. However, the fluorescent signals coming from the

silica beads that adhered to cancer cells were noticeably

less different from that of the immortal cells when com-

pared to that of either normal or infected cells (see, the

values of probability and q-value in Table S4; further

details of the statistical analysis are shown in the Supple-

mentary Materials (Tables S2–S4).

To summarize, our results show a statistically significant

(at p \ 0.01) difference between all types of cells except

normal-infected consisting of normal and infected and a

group of immortal and cancer cells. This implies that the

cell surface changes substantially when cells become

immortal in its progression to cancer.

Discussion

Here we study the change of the surface of human cervical

epithelial cells during this progression to cancer, from

normal, to HPV-infected, to immortal, and then to the

carcinoma stage. Such a change has previously been

reported when comparing normal and cancerous epithelial

cervical cells [29, 31, 32], and was attributed to the change

of microvilli content of the cellular brush. Physical

adsorption of fluorescent silica beads is used to monitor the

change of cell surface, the method described in Ref. [31].

Fig. 2 (Color online)

2009110 lm2 optical images of

normal, infected, immortal, and

cancer cells with fluorescent

silica particles attached. The

cells are seen as an almost

continuous (green) background,

and the particles are the bright

spots (red)

Cell Biochem Biophys (2012) 63:109–116 113

123

Visual inspection of the optical images (Fig. 2) shows that

the number of particles adsorbed on the cancer and

immortal cell surfaces is typically more than that on the

normal and infected cell surface. The quantitative mea-

surements of fluorescent signal coming from the fluores-

cent silica beads adherent to the cell surface, Fig. 3a),

confirms this observation statistically with the confidence

level of 0.01.

Figure 3b shows a noticeable variability of the fluores-

cent signals collected from different human subjects. While

this variability does not change the conclusion of the

alteration of the cell surface starting from the stage of

immortalization, this implies the need for studying more

human subjects if this method is going to be used for

diagnostic purposes.

To determine whether the presence of E6 and E7 genes

alters the cell surface (and consequently, the number of

silica particles adsorbed to the cell surface), we compared

the fluorescence signal coming from the silica beads

adhered to normal and infected cells. Because the fluo-

rescence signals from normal and infected cells were

statistically similar (the difference was statistically insig-

nificant at the confidence level p \ 0.01), one could con-

clude that the infected cells have surface characteristics

similar to normal cells. From this we can conclude that just

the presence of E6 and E7 genes is not sufficient to change

the cell surface.

It was reported that the two genes E6 and E7 have to act

synergistically to induce cell immortalization [4]. It was

speculated that E7 can prolong the lifespan of cells and

therefore, supposedly induce overgrowth of cells. How-

ever, E7 can also induce apoptosis [39]. E6 inhibits the

apoptosis induced by E7, accentuating the effects of E7 on

immortalization [40]. Synergistic action of the oncogenes

is a progressive time-dependent process. From this we can

conclude that the synergistic action of both E6 and E7

genes alone is not sufficient to change the cell surface. One

can see that the fluorescent signal is stronger in the case of

immortal cells, and getting closer to cancerous cells. It is

also in agreement with in vivo results of Ref. [41] in which

it was shown that immortal cells of similar passages used

here behaved more as cancerous cells. In addition, the

observed difference between normal and cancer cells is in

agreement with the data reported previously [29].

To amplify, we showed that the cell surface changes

substantially at the stage of immortalization. This implies

direct correlation between the increased adhesion and

expression of both E6 and E7 genes. In the light of results

of Ref. [29], it presumably means that the cellular brush

(microvilli, microridges, glycocalyx, etc.) alters substan-

tially when cells become immortal. Second, our results

showed that the physical labeling of cervical epithelial cells

with fluorescent silica beads could be suitable for dis-

crimination of both immortal and cancerous cervical cells

in a culture dish. This demonstrates the significance of

physical properties of the cell surface for the development

of clinical methods for early detection of cervical cancer,

even at the stage of immortalized premalignant cells. Such

detection would represent a substantial departure from the

traditional biochemically specific labeling methods.

Acknowledgments Funding for this study from the National Sci-

ence Foundation NSF CBET 0755704 and ARO W911NF-05-1-0339

(I.S.) and the National Cancer Institute 1R15CA126855-01 (C.W.) are

acknowledged. Human tissue was obtained from the Cooperative

Human Tissue Network.

Conflict of interest The authors have no conflicts of interest to

declare.

Fig. 3 The fluorescent signals collected from silica beads adhered to

cells taken from three healthy individuals, three cell stains infected

with HPV-16 virus (derived from cells of three healthy individuals),

three immortal cell lines (derived from three cell stains infected with

HPV-16 virus), and malignant cells from three cancer patients. a The

averaged fluorescent signal for all human subjects and one standard

deviation are shown in the graph. b The average (middle line in the

box), one standard deviation (the box) and minimum/maximum

(error-bar markers) are shown for each human subject separately

114 Cell Biochem Biophys (2012) 63:109–116

123

References

1. Walboomers, J. M., Jacobs, M. V., Manos, M. M., Bosch, F. X.,

Kummer, J. A., Shah, K. V., et al. (1999). Human papillomavirus

is a necessary cause of invasive cervical cancer worldwide.

Journal of Pathology, 189, 12–19.

2. Sprintz, M. (2004). Editorial: Nanotechnology for advanced

therapy and diagnosis. Biomedical Microdevices, 6, 101–103.

3. Wright, T. C., Kurman, R. J., & Ferenczy, A. (1994). In R. J. Kurman

(Ed.), Precancerous lesions of the cervix in Blaustein’s pathology ofthe female genital tract (pp. 229–278). New York: Springer-Verlag.

4. Song, S., Liem, A., Miller, J. A., & Lambert, P. F. (2000). Human

papillomavirus types 16 E6 and E7 contribute differently to

carcinogenesis. Virology, 267, 141–150.

5. Cooper, K., Herrington, C. S., Lo, E. S., Evans, M. F., & McGee,

J. O. (1992). Integration of human papillomavirus types 16 and

18 in cervical adenocarcinoma. Journal of Clinical Pathology,45, 382–384.

6. Daniel, B., Rangarajan, A., Mukherjee, G., Vallikad, E., &

Krishna, S. (1997). The link between integration and expression

of human papillomavirus type 16 genomes and cellular changes

in the evolution of cervical intraepithelial neoplastic lesions.

Journal of General Virology, 78(Pt 5), 1095–1101.

7. von Knebel Doeberitz, M., Oltersdorf, T., Schwarz, E., &

Gissmann, L. (1988). Correlation of modified human papilloma

virus early gene expression with altered growth properties in

C4–1 cervical carcinoma cells. Cancer Research, 48, 3780–3786.

8. Huibregtse, J. M., Scheffner, M., & Howley, P. M. (1991).

A cellular protein mediates association of p53 with the E6

oncoprotein of human papillomavirus types 16 or 18. EMBOJournal, 10, 4129–4135.

9. Werness, B. A., Levine, A. J., & Howley, P. M. (1990). Asso-

ciation of human papillomavirus types 16 and 18 E6 proteins with

p53. Science, 248, 76–79.

10. Dyson, N., Guida, P., Munger, K., & Harlow, E. (1992).

Homologous sequences in adenovirus E1A and human papillo-

mavirus E7 proteins mediate interaction with the same set of

cellular proteins. Journal of Virology, 66, 6893–6902.

11. Jones, D. L., Thompson, D. A., & Munger, K. (1997). Destabi-

lization of the RB tumor suppressor protein and stabilization of

p53 contribute to HPV type 16 E7-induced apoptosis. Virology,239, 97–107.

12. Nuovo, J., Melnikow, J., & Howell, L. P. (2001). New tests for

cervical cancer screening. American Family Physician, 64,

780–786.

13. Monsonego, J., Bosch, F. X., Coursaget, P., Cox, J. T., Franco, E.,

Frazer, I., et al. (2004). Cervical cancer control, priorities and

new directions. International Journal of Cancer, 108, 329–333.

14. Belinson, J. L., Pretorius, R. G., Zhang, W. H., Wu, L. Y., Qiao,

Y. L., & Elson, P. (2001). Cervical cancer screening by simple

visual inspection after acetic acid. Obstetrics and Gynecology,98, 441–444.

15. Bhatla, N., Mukhopadhyay, A., Joshi, S., Kumar, A., Kriplani, A.,

Pandey, R. M., et al. (2004). Visual inspection for cervical cancer

screening: Evaluation by doctor versus paramedical worker.

Indian Journal of Cancer, 41, 32–36.

16. Marcelli, M., Ittmann, M., Mariani, S., Sutherland, R., Nigam, R.,

Murthy, L., et al. (2000). Androgen receptor mutations in prostate

cancer. Cancer Research, 60, 944–949.

17. Scarcigilia, L., Compagnone, D., Fedrici, G., & Palleschi, G.

(1998). Electrochemical probe for polyamines detection in bio-

logical fluids. Analysis, 26, 219–222.

18. Adamek, H. E., Albert, J., Breer, H., Weitz, M., Schilling, D., &

Riemann, J. F. (2000). Pancreatic cancer detection with magnetic

resonance cholangiopancreatography and endoscopic retrograde

cholangiopancreatography: A prospective controlled study. Lan-cet, 356, 190–193.

19. Fodor, S. P., Rava, R. P., Huang, X. C., Pease, A. C., Holmes, C.

P., & Adams, C. L. (1993). Multiplexed biochemical assays with

biological chips. Nature, 364, 555–556.

20. Weissleder, R., Tung, C. H., Mahmood, U., & Bogdanov, A., Jr.

(1999). In vivo imaging of tumors with protease-activated near-

infrared fluorescent probes. Nature Biotechnology, 17, 375–378.

21. Ramanujam, N., Mitchell, M. F., Mahadevan-Jansen, A., Thomsen,

S. L., Staerkel, G., Malpica, A., et al. (1996). Cervical precancer

detection using a multivariate statistical algorithm based on laser-

induced fluorescence spectra at multiple excitation wavelengths.

Photochemistry and Photobiology, 64, 720–735.

22. Drezek, R. A., Collier, T., Brookner, C. K., Malpica, A., Lotan, R.,

Richards-Kortum, R. R., et al. (2000). Laser scanning confocal

microscopy of cervical tissue before and after application of

acetic acid. American Journal of Obstetrics and Gynecology, 182,

1135–1139.

23. Pitris, C., Goodman, A., Boppart, S. A., Libus, J. J., Fujimoto, J. G.,

& Brezinski, M. E. (1999). High-resolution imaging of gyneco-

logic neoplasms using optical coherence tomography. Obstetricsand Gynecology, 93, 135–139.

24. Mitchell, M. F., Cantor, S. B., Ramanujam, N., Tortolero-Luna, G.,

& Richards-Kortum, R. (1999). Fluorescence spectroscopy for

diagnosis of squamous intraepithelial lesions of the cervix.

Obstetrics and Gynecology, 93, 462–470.

25. Bui, J. D., Zelles, T., Lou, H. J., Gallion, V. L., Phillips, M. I., &

Tan, W. (1999). Probing intracellular dynamics in living cells

with near-field optics. Journal of Neuroscience Methods, 89,

9–15.

26. Tan, W., Parpura, V., Haydon, P. G., & Yeung, E. S. (1995).

Neurotransmitter imaging in living cells based on native fluo-

rescence detection. Analytical Chemistry, 67, 2575–2579.

27. Fung, J. C., Marshall, W. F., Dernburg, A., Agard, D. A., &

Sedat, J. W. (1998). Homologous chromosome pairing in Dro-

sophila melanogaster proceeds through multiple independent

initiations. Journal of Cell Biology, 141, 5–20.

28. Bornhop, D. J., Griffin, J. M., Goebel, T. S., Sudduth, M. R., Bell,

B., & Motamedi, M. (2003). Luminescent lanthanide chelate

contrast agents and detection of lesions in the hamster oral cancer

model. Applied Spectroscopy, 57, 1216–1222.

29. Iyer, S., Gaikwad, R. M., Subba-Rao, V., Woodworth, C. D., &

Sokolov, I. (2009). Atomic force microscopy detects differences

in the surface brush of normal and cancerous cells. NatureNanotechnology, 4, 389–393.

30. Sokolov, I., Iyer, S., & Woodworth, C. D. (2006). Recovery of

elasticity of aged human epithelial cells in vitro. Nanomedicine,2, 31–36.

31. Iyer, S., Woodworth, C. D., Gaikwad, R. M., Kievsky, Y. Y., &

Sokolov, I. (2009). Towards nonspecific detection of malignant

cervical cells with fluorescent silica beads. Small, 5, 2277–2284.

32. Gaikwad, R. M., Dokukin, M. E., Iyer, K. S., Woodworth, C. D.,

Volkov, D. O., & Sokolov, I. (2011). Detection of cancerous

cervical cells using physical adhesion of fluorescent silica parti-

cles and centripetal force. Analyst, 136, 1502–1506.

33. Woodworth, C. D., Doniger, J., & DiPaolo, J. A. (1989).

Immortalization of human foreskin keratinocytes by various

human papillomavirus DNAs corresponds to their association

with cervical carcinoma. Journal of Virology, 63, 159–164.

34. Woodworth, C. D., Cheng, S., Simpson, S., Hamacher, L., Chow,

L. T., Broker, T. R., et al. (1992). Recombinant retroviruses

encoding human papillomavirus type 18 E6 and E7 genes stim-

ulate proliferation and delay differentiation of human keratino-

cytes early after infection. Oncogene, 7, 619–626.

35. Cho, E. B., Volkov, D. O., & Sokolov, I. (2010). Ultrabright

fluorescent mesoporous silica nanoparticles. Small, 6, 2314–2319.

Cell Biochem Biophys (2012) 63:109–116 115

123

36. Sokolov, I., Kievsky, Y. Y., & Kaszpurenko, J. M. (2007). Self-

assembly of ultrabright fluorescent silica particles. Small, 3,

419–423.

37. Naik, S. P., & Igor, S. (2008). Ultra-bright fluorescent silica

particles: physical entrapment of fluorescent dye rhodamine 640

in nanochannels. In R. Nagarajan (Ed.), Nanoparticles: Synthesis,stabilization, passivation and functionalization (pp. 214–224).

London: ACS.

38. Han, M., Gao, X., Su, J. Z., & Nie, S. (2001). Quantum-dot-

tagged microbeads for multiplexed optical coding of biomole-

cules. Nature Biotechnology, 19, 631–635.

39. Pan, H., & Griep, A. E. (1994). Altered cell cycle regulation in

the lens of HPV-16 E6 or E7 transgenic mice: Implications for

tumor suppressor gene function in development. Genes andDevelopment, 8, 1285–1299.

40. Pan, H., & Griep, A. E. (1995). Temporally distinct patterns of

p53-dependent and p53-independent apoptosis during mouse lens

development. Genes and Development, 9, 2157–2169.

41. Woodworth, C. D., Waggoner, S., Barnes, W., Stoler, M. H., &

DiPaolo, J. A. (1990). Human cervical and foreskin epithelial

cells immortalized by human papillomavirus DNAs exhibit dys-

plastic differentiation in vivo. Cancer Research, 50, 3709–3715.

42. Woodworth, C. D., McMullin, E., Iglesias, M., & Plowman, G. D.

(1995). Interleukin 1 alpha and tumor necrosis factor alpha

stimulate autocrine amphiregulin expression and proliferation of

human papillomavirus-immortalized and carcinoma-derived cer-

vical epithelial cells. Proceedings of the National Academy of theSciences of the United States of America, 92, 2840–2844.

43. Dokukin, M. E., Guz, N. V., Gaikwad, R. M., Woodworth, C. D.,

& Sokolov, I. (2011). Cell surface as a fractal: Normal and

cancerous cervical cells demonstrate different fractal behavior of

surface adhesion maps at the nanoscale. Physical Review Letters,107, 028101.

44. Katki, H. A., & Wentzensen, N. (2012). How might HPV testing

be integrated into cervical screening? The lancet Oncology, 13,

8–10.

116 Cell Biochem Biophys (2012) 63:109–116

123