Citation
Hazelbag, S. (2006, February 2). Transforming Growth Factor beta-1 in
cervical cancer. Retrieved from https://hdl.handle.net/1887/4320
Version: Corrected Publisher’s Version
License: Licence agreement concerning inclusion of doctoralthesis in the Institutional Repository of the University of Leiden
Downloaded from: https://hdl.handle.net/1887/4320
C E R V I C A L
C A R C I N O M A
C E L L S
Suzanne Hazelbag1,2, Gert Jan Fleuren1, J.J. Baelde1, Ed Schuuring1, Gemma G. Kenter2 and Arko Gorter1,
1 Dept of Pathology, Leiden University Medical Center, Leiden, the Netherlands 2 Dept of Obstetrics and Gynaecology, Leiden University Medical Center, Leiden, the Netherlands
A
B S T R A C TObjective: in patients with cervical carcinoma, the presence of cytokines produced by TH2-cells, and the presence of an eosinophilic inflammatory infiltrate has been associated with a less effective immune response and tumor progression. In the present study, we have investigated the cytokine profile of cervical carcinoma cells. In addition, we have measured whether differences in cytokine profile are present between normal and malignant cervical epithelial cells.
Methods: for this purpose we have determined the mRNA expression pattern of twenty relevant cytokines by RT-PCR and Southern blotting in three normal primary cervical epithelial cell cultures (NPE) and ten cervical cancer cell lines (CCCL).
Results: TGF-β1, IL-4, IL-12p35 and IL-15 were produced by all CCCL and NPE. TNF-α, IL-10, IL-5 and RANTES, were present in most NPE, but not in any of the CCCL. MCP-1 was expressed in all CCCL but only in one NPE. The presence of the anti-inflammatory cytokine TGF-β1 in cervical carcinomas was confirmed by RNA in situ hybridization on tissue sections of carcinomas from which the CCCL originated.
I
N T R O D U C T I O NCervical cancer is the second leading cause of cancer death in women worldwide.1 It is well established that chronic infection of keratinocytes in the uterine cervix by human papilloma viruses (HPV), in particular the oncogenic types (e.g. HPV type 16 and 18), plays an important role in the pathogenesis of cervical cancer. In about 95 % of all biopsies derived from cancer of the cervix throughout the world HPV was detected.2 The host immune response to HPV infection is thought to be a crucial factor in the clearance of the virus. Cell-mediated immune responses are believed to be essential in controlling both HPV-infections and HPV-related neoplasm’s.3 This is supported by the observation that an ineffective cellular immune response, as in immunocompromised individuals like transplant recipients and patients infected with human immunodeficiency virus (HIV), is associated with an increased incidence of HPV-related disorders.4-6
Important immunological mediators of cell mediated defenses against tumors are cytokines. T helper (TH) cells can be distuinghuised into TH1 and TH2 cells by the type of cytokines they produce. TH1 cytokines are pro-inflammatory or immunostimulatory cytokines that boost the cellular immune response, whereas TH2 cytokines are anti-inflammatory or immuno-inhibitory cytokines with the capacity to subvert the cellular immune response. A shift towards cytokines produced by TH2 cells has been associated with a less effective immune response in a number of cancer types.7,8 In cervical carcinoma biopsies a decline of gamma-interferon (IFN-γ), interleukin-12 (IL-12) and monocyte chemoattractant protein (MCP)-1 mRNA expression was observed in comparison with normal cervix tissue.9-12
Many of the cytokines are presumed to be produced by inflammatory cells that are a present in a substantial amount in the tumor samples. However, Woodworth et al. have shown that cervical carcinoma cells as well as normal keratinocytes can produce some cytokines.13 An other interesting observation is the presence of a high number of eosinophilic granulocytes in 5-40% of the cervical tumors.14-16 The presence of an eosinophilic infiltrate in cervical cancer has been associated with a less effective immune response.17
cervical carcinoma cells produce a significant number of immunomodulatory cytokines. To exclude the influence of the presence of inflammatory cells in tissue samples, we have investigated the expression of twenty different cytokines in ten different CCCL. Furthermore, we have investigated whether differences in cyto-kine profile are present between normal and malignant cervical epithelial cells. Finally , to investigate the relevance of the expressed cytokines by CCCL, we have determined whether in tissue sections of cervical carcinomas from which the CCCL originated, cytokine expression (e.g. TGF-β1 ), could be detected.
M
A T E R I A L A N DM
E T H O D SCells and Cell lines
NPE could be cultured for 2 to 4 passages before cells would stop growing and die. Cultured NPE contained less than one percent inflammatory cells and were morphologically devoid of contaminating fibroblasts.
CSCC and CC cell lines were grown in serum-free keratinocyte medium sup-plemented with 5 μg/L rhEGF and 50 mg/L bovine pituitary extract, whereas SiHa, HeLa and CaSki were cultured in Dulbecco’s MEM (DMEM) containing 10 % heat FCS, 50 U/ml penicillin-50 μg/ml streptomycin and 2 mM glutamine (DMEM complete medium), all purchased from Life Technologies (Rockville, MD, USA). Cervical cancer tumor cell suspensions were obtained from tumor specimens of patients undergoing hysterectomy because of cervical carcinoma. Tumor speci-mens were obtained immediately after surgery. The specimen was cut into 1 mm3 pieces and washed twice in DMEM. Subsequently, per 1 g of tissue, 5 ml FCS-free DMEM medium containing 4 mg/ml collagenase Ia (Sigma) and 0.002 % DNAse (Sigma) was added. The tissue was incubated in this medium for 1 h at 370 C. The acquired cell suspensions were filtered over a 100 μm nylon gauze (Verseidag-Industrietextilien GmbH, Kempen, FRG) and washed twice with DMEM complete medium. Single cell suspensions were frozen (DMSO 10 %/ FCS 60 %/ complete DMEM 30 %) and stored in liquid nitrogen until use.
The bladder carcinoma cell line 563723 as kindly provided by Roel de Paus (Dept of Experimental Hematology, LUMC). Dermal fibroblasts were a kind gift from Maya Ponec (Dept. of Dermatology, LUMC). Fibroblasts were stimulated with tumor necrosis factor-alpha (TNF-α) (30 ng/ml) and IL-4 (5 ng/ml) for 24 h.24 Peripheral blood mononuclear cells (PBMC) were isolated from buffy coats of healthy donors using Ficoll-Hypaque gradient sedimentation (1.077 g/cm3, 1000 g, 20 min, 200 C). PBMC were stimulated with phytohemagglutinin (PHA) (150 μg/L x 106 cells/ml; Difco, Detroit, MI) and recombinant human IL-2 (rhIL-2) (100 U/ml, Eurocetus, Amsterdam, NL) in RPMI 1640 (Life Technologies) for 24 h.23 The bladder carcinoma cell line 5637, dermal fibroblasts and PBMC were used as positive controls to detect specific cytokine products in the PCR as described below.
RT-PCR
Madison, WI, USA) in a total volume of 20 μl. Samples were incubated at 37 0 C for 1 h. In parallel, reverse transcription of each sample was performed in the absence of RT to check for amplification of genomic DNA sequences.
For PCR-mediated amplification of cDNA, 1 μl of cDNA solution was added to 5 μl PCR buffer (1 mM Tris pH 8.4, 5 mM KCl, 0.006% BSA), 2 mM MgCl2, 0.2 mM dNTPs, 0.5 pmol sense and 0.5 pmol antisense primer, 1 U AmpliTaq DNA polymerase (PE Biosystems, Foster City, CA) in a total volume of 50 μl. Primers for IL-1-α, IL-1-β, IL-2, IL-4, IL-5, IL-6, IL-8, granulocyte macrophage-colony stimulating factor (GM-CSF), IFN-γ, TNF-α, IL-10, IL-12 p35 and IL-12 P40,11 IL-15,25 IL-16,26 monocyte chemoattractant protein-1 (MCP-1), MCP-3, RANTES27 and eotaxin28 were selected as previously described. Primers for TGF-β
TA B L E 1 - Primer pairs and internal oligoprobes.
Human Annealing Product
gene Temp., ˚C Length, bp Sequence
IL-1-α 55 491 sense: 5’-ACCAACCAGTGCTGCTGAAG
antisense: 5’-CATAGTCAGTAGCTCTGGTC probe: 5’-GACCAACCTCCTCTTCTTCTGGGA
IL-1-β 55 443 sense: 5’-GATAAGCCCACTCTACAGCT
antisense: 5’-ATTGGCCCTGAAAGGAGAGA probe: 5’-TGATGGACAGGAGATCCTCTTAGC
IL-2 55 299 sense: 5’-CAACGTAATAGTTCTGGAAC
antisense: 5’-TAGGGCTTACAAAAAGAATC probe: 5’- GAATATGCTGATGAGACAGCAACC
IL-4 55 278 sense: 5’-ACACCTATTAATGGGTCTCA
antisense: 5’-CAACTGAGAAGGAAACCTTC probe: 5’- GCTCCGGCAGTTCTACAGCCACCA
IL-5 55 367 sense: 5’-AGCTCCAAGAGCTAGCAAACT
antisense: 5’-GCAAAGTGTCAGTATGCCTG probe: 5’-CACCAACTGTGCACTGAAGA
IL-6 55 819 sense: 5’-GTACCCCCAGGAGAAGATTC
antisense: 5’-CAAACTGCATAGCCACTTTC probe: 5’-CAAAGATGTAGCCGCCCCACACAG
IL-8 55 585 sense: 5’-GCTTTCTGATGGAAGAGAGC
antisense: 5’-TGTGGATCCTGGCTAGCAGA probe: 5’- TGTGGGTCTGTTGTAGGGTTGCCA
IL-10 60 564 sense: 5’-GAAAACAAGAGCAAGGCCGTGGAG
antisense: 5’-CCCAGAGACAAGATAAATTAGAGG probe: 5’-GAAGATACGAAACTGAGACATCAGG IL-12-p35 60 372 sense: 5’-CATAACTAATGGGAGTTGCCTGGC
antisense: 5’-AACGGTTTGGAGGGACCTCG probe: 5’- GCTTCTGATGGATCCTAAGAGGCAG IL-12-p40 60 444 sense: 5’-GGCCAGTACACCTGTCACAAA
antisense: 5’-TGATGATGTCCCTGATGAAGAAGC probe: 5’-TACACTCTCTGCAGAGAGAGTCAG-AGG
IL-15 55 430 sense: 5’-GCCTTCATGGTATTGGGAAC
antisense: 5’-GAATCAATTGCAATCAAGAAGTG probe: 5’-GAGTCCGGAGATGCAAGTATTCATG
IL-16 60 347 sense: 5’-ATGCCCGACCTCAACTCCTC
antisense: 5’-CTCCTGATGACAATCGTGAC probe: 5’- GACAAGCCCCTCACCATTAACAGGA
TNF-α 55 468 sense: 5’-GCCTGTAGCCCATGTTGTAG
antisense: 5’-TTGGGAAGGTTGGATGTTCG probe: 5’-TGCCATCAGAGGGCCTGTACCTCA
IFN-γ 55 634 sense: 5’- TCCAAAAGAGTGTGGAGACC
antisense: 5’- AAGCACTGGCTCAGATTGCA probe: 5’- GGGAAGCGAAAAAGGAGTCAGA TGF-β1 59 473 sense: 5’- AGCGACTCGCCAGAGTGGTTATCTT antisense: 5’- AATACGACCAACATGTCCCGGCCT probe: 5’- ATGGCATGAACCGGCCTTTCCTGCT MCP-1 61 297 sense: 5’- GCTCATAGCAGCCACCTTCATTC antisense: 5’- TGCAGATTCTTGGGTTGTGGAG probe: 5’- CATTGTGGCCAAGGAGATCTGTGCTGAC MCP-3 55 276 sense: 5’- CCCAGGGGCTTGCTCAG antisense: 5’- CATGGCTTGTTTTCAGTTCAGTCA probe: 5’- GCCACTGTCCCCGGGAAGCTGTAAT Eotaxin 62 207 sense: 5’- CCCAACCACCTGCTGCTTTAACCTG
antisense: 5’- GCTTTGGAGTTGGAGATTTTTG
probe: 5’- GGAGAATCACCAGTGGCAAATGTCCCCAG
RANTES 62 618 sense: 5’- CATCCTCATTGCTACTGCCCTCTG
antisense: 5’- CGGGTTCACGCCATTCTCCT probe: 5’- CCCAGAGAAGAAATGGGTTCGGGAG
GM-CSF 55 423 sense: 5’- CAGAAATGTTTGACCTCCAG
RNA in Situ Hybridization
The in situ hybridization was performed on paraffin-embedded sections of the tumors from which cell lines CC-10 and CC-11 originated from and carried out as formerly described.29 In short, after pretreatment the sections were hybridized with 10 ng TGF-β riboprobe per slide during 16 h at 620 C. Subsequently, sections were washed in 2x standard saline solution citrate (SSC) with 50% formamide at 500 C, then in 0.1x SSC with 20 mM β-mercaptoethanol at 620 C, and finally treated with 2 U/ml ribonuclease (RNAse) T1 (Roche) in 2x SSC plus 1 mM ethyle nediaminetetraacetic acid (EDTA) at 370 C. The immunodetection of digoxigenin-labeled hybrids was done using nitro blue tetrazolium (NBT) as chromogen and bicholylindolyl phosphate (BCIP) as coupling agent (Roche). The sense probes were included as negative controls, and did not show staining.
R
E S U L T STo determine the cytokine expression profile of cervical carcinoma cells, we tested ten CCCL for the expression of twenty cytokines. In addition, to determine differences in expression patterns between normal and cancer cells we have compared the cytokine expression patterns of CCCL with NPE.
Cytokines were divided in three groups: anti-inflammatory cytokines, pro-inflammatory cytokines and growth or chemotactic cytokines. The selection of the cytokines was based on their known immunomodulatory properties on cervical cancer cells, antigen presenting cells or effector cell populations (NK cells, T cells or eosinophilic granulocytes). With regard to the effector cell populations, a striking feature of cervical carcinomas is the presence of a high proportion of eosinophilic granulocytes. The presence of these cells in cervical carcinoma has been associated with a less effective immune response.
Cytokine profiles of CCCL and NPE Anti-inflammatory cytokines
Of the anti-inflammatory cytokines, only IL-4 and TGF-β1 mRNA were
TA B L E 2 - Cytokine profiles of normal and malignant cervical epithelial cells and tumour cells.
Cytokines NPE CCCL tumor cell suspension
(n=3) (n=10) (n=6) anti-inflammatory IL-4 3 4 5 IL-5 2 0 4 IL-10 2 0 6 TGF-β1 3 10 6 pro-inflammatory IL-2 1 1 6 IL-15 3 10 6 IL-12p40 1 2 6 IL-12p35 3 9 6 IFN-γ 0 1 6 TNF-α 2 0 4 GM-CSF 3 3 5 growth factors IL-1-α 3 9 5 IL-1-β 3 10 6 IL-6 3 8 6 chemotactic factors IL-8 3 9 6 IL-16 1 1 6 MCP-1 1 10 6 MCP-3 0 1 6 Eotaxin 0 1 6 RANTES 3 0 5 Pro-inflammatory cytokines
Of the pro-inflammatory cytokines, IL-15 and IL-12p35 mRNA were detected in almost all CCCL, whereas IL-2, IL-12p40 and IFN-γ were occasionally detected (Table 2). TNF-α was not detected in any of the CCCL investigated. When the expression pattern of the pro-inflammatory cytokines was compared between CCCL and NPE, IL-15 and IL-12p35 were detected in all CCCL and NPE. Occasional expression of IL-2 and IL-12p40 was found in both CCCL and NPE. Interestingly, the cervical carcinoma cell line that expressed IL-2 was the only cell line that expressed IL-15 weakly (CSCC-1, Fig.1 and 2). TNF-α was expressed in the majority of the NPE, but not detected in any of the CCCL.
Growth- and chemotactic cytokines
FI G U R E 1 - Expression of anti-inflammatory cytokines mRNA in CCCL.
Abbreviations; CSCC-1(1), CSCC-7(7), CC-8(8), CC-10a(10a), CC-10b(10b), CC-11-(11-), CC-11+(11+), SiHa(S), HeLa(H), CaSki(C), a NPE (n), a primary tumor cell suspension (s), a positive control for each cytokine (PBMC or the bladder carcinoma cell line 5637 (TGF-β1)) (c) and a control for genomic DNA (d). Next to each specimen (right side) a RT negative sample of
each specimen was applied to distinguish between copy (c) and genomic (g) DNA products. Specific cDNA products were visualized as a band of the appropriate length after Southern blotting. Expression of HPRT was measured to estimate the quantity of the different mRNAs applied in each lane. Note that bands with the appropriate length for IL-5 are present in c and n, bands with the appropriate length for IL-10 are present in c, s and n. Other bands generally represent aspecific gDNA products.
FI G U R E 2 - Expression of proinflammatory cytokines mRNA in CCCL.
Abbreviations: CSCC-1(1), CSCC-7(7), CC-8(8), CC-10a(10a), CC-10b(10b), CC-11-(11-), CC-11+(11+), SiHa(S), HeLa(H), CaSki(C), a NPE (n), a primary tumor cell suspension (s), a positive control for each cytokine (PBMC) (c) and a control for ge-nomic DNA (d). Next to each specimen (right side) a RT negative sample of each specimen was applied to distinguish between copy (c) and genomic (g) DNA cytokine products. Specific cDNA products were visualized as a band of the appropriate length after Southern blotting. Expression of HPRT was measured to estimate the quantity of the different mRNAs applied in each lane. Note that bands with the appropriate length for IL-2 are present in 1, s and c, bands with the appropriate length for IL-12p40 are present in 1, 7, s and c and bands with the appropriate length for IFN-g are present in 1, s and c. Other bands generally represent aspecific gDNA products.
Cervical carcinomas
To clarify the in vivo relevance of cytokine mRNA expression by CCCL, we selected the anti-inflammatory cytokine TGF-β1 and measured whether this cytokine was also expressed by cervical cancer cells in situ.
FI G U R E 3 - TGF-β1 expression in cervical carcinomas. mRNA was detected by RNA in situ hybridization with
an anti-sense riboprobe for TGF-β1 as described in Materials and Methods. TGF-β1 is visualized by a blue color. (Original magnification: x 200.)
Fig. 3a. Expression of TGF-b1 mRNA in the cytoplasm of cervical cancer cells of the primary cervical tumor (Tu), from which the cell line CC-11 originates. Note inflammatory cells in the tumor infiltrate also expressing TGF-b1 mRNA (arrow).
Fig. 3b. Negative control (sense riboprobe).
D
I S C U S S I O NIn the present study the cytokine mRNA expression profile of cervical carcinoma cells was examined. In addition, in order to detect differences in expression pattern, the cytokine expression pattern of normal cervical epithelial cells and cervical cancer cells was compared. Only few studies have examined the cytokine expression pattern of pure cervical tumor cells.13 Most studies have investigated the cytokine expression patterns in cervical tumor biopsies.9-11 In these cases, cytokines produced by inflammatory cells and stromal cells are also measured. To substantiate this statement, we have also measured the cytokine expression pattern of six primary tumor cell suspensions. As expected, we observed that all the tumor cell suspensions, contrary to the carcinoma cell lines, expressed mRNA for virtually all the tested cytokines (Table II). This illustrates the influence of inflammatory cells and stromal cells on the cytokine profiles determined in tumor biopsies and the impossibility to distinguish between the contribution of the carcinoma cells and other cells to the cytokine profile, using RT-PCR techniques. Therefore it is important to first establish a cytokine profile from CCCL.
When the cytokine profile of CCCL and NPE was measured we observed that of the anti-inflammatory cytokines, IL-4 mRNA expression was found in only a number of CCCL as compared to all NPE. These findings are in contrast with those of other investigators,11,19,13 who detected elevated expression of IL-4 in carcinoma biopsies. As discussed previously, since TH2 cells are an important source of IL-4 production,31 the elevated expression in these studies is probably the result of an increased IL-4 production by inflammatory (TH2) cells.
TGF-β1 mRNA expression was detected in CCCL, in situ in cervical cancer cells and NPE. The expression of TGF-β1 by both CCCL and cervical cancer cells in situ suggests that cervical cell lines are a suitable model for studying cytokine expression pattern in cervical tumor cells (Fig. 2 and 3). The expression of TGF-β1 by NPE is consistent with the observations of Al Saleh et al.30 and de Gruijl et al..11 TGF-β1 has been shown to inhibit cell proliferation.32 Normal (cervical) epithelial cells may modulate their growth by regulating their TGF-β1 production.
Production of TGF-β1 by cervical cancer cells may have important immuno-modulatory consequences for the local tumor environment. It has been shown that TGF-β1 inhibits CTL generation18.33 and that TGF-β
1 is the major factor involved in the inhibition of generation of lymphokine activated killer (LAK) cells.10 In addition, TGF-β
1 has been shown to diminish the production of immunostimulatory cytokines, such as TNF-α and IFN-γ.18 TGF-β
reported to stimulate the expression of the laminin receptor on Langerhans’ cells. The laminin receptor is capable of facilitating cellular uptake and expression of HPV.34 Langerhans’ cells expressing E7 have been shown to be poor inducers of T help.35 TGF-β
1 also enhances IL-10 production by macrophages, which is known to be a potent immunosuppressive cytokine as well.36,37 Indeed, all six primary tumor cell suspensions tested expressed both IL-10 and TGF-β1 mRNA, while none of the carcinoma cell lines expressed IL-10 (Table 2). All tumor cell suspensions contained a varying percentage of other than tumor cells.38 As previously shown, this supports the idea, that cervical carcinoma cells via production of TGF-β1 upregulate IL-10 production in the macrophages of the tumor infiltrate. Thus the (over) production of TGF-β1 (and IL-10) could both reduce the level of antigen presentation by Langerhans’ cells as well as reduce the cytotoxic potential of the cytotoxic T cells.
As previously stated TGF-β1 has been shown to inhibit cell proliferation.32 Despite the production of TGF-β1 the cervical cancer cells themselves do frequently not respond to its growth inhibitory properties.39-41 This suggests that cervical carcino-mas benefit from loss of sensitivity for TGF-β1. Loss of sensitivity for TGF-β1 may provide the cervical carcinoma cells with both a growth advantage (loss of growth inhibition) and may allow increased local TGF-β1 concentration, due to sustained TGF-β1 production, thus limiting an efficient anti-tumor immune response. The underlying processes explaining this loss of sensitivity for TGF-β1 of cervical cancer cells are currently unknown. Occasional mutations in TGFβ-receptors have been described for cervical carcinoma, partly explaining their lack of sensitivity to TGF-β1. 40,42,43
The observation of Santin et al.,44 that TGF-β
1 expression was only detected in adenocarcinoma and not in squamous carcinoma, was not confirmed by our results in CCCL. Both squamous carcinoma cell lines and adeno- or adenosquamous carcinoma cell lines expressed similar amounts of TGF-β1 mRNA.
expression on tumor cells and promotes cytotoxicity towards tumor cells.46 GM-CSF also induces differentiation and maturation of Langerhans’ cells. In addition. GM-CSF induces production of the pro-inflammatory cytokine IL-12 by dendritic cells.47 Thus production of GM-CSF may increase the immunogenicity of tumors and induce anti-tumor immunity.48,49
Although all CCCL and NPE expressed IL12p35 mRNA, only occasional expres-sion of IL-12p40 was detected. We have not determined whether the simultaneous expression of IL-12p35 and IL-12p40 mRNA results in the production of biologi-cally functional IL-12. However interestingly, the few CCCL that produced both subunits of IL-12 were the same CCCL that still produced GM-CSF. The production of IL-12p40 by these CCCL was not due to HPV infection alone, since in our study a HPV negative NPE expressed IL-12p40 as well. It could be very well possible that GM-CSF can induce IL-12 production in tumor cells as well as dendritic cells. This raises the question if patients with GM-CSF/IL-12 producing tumors have better survival rates than patients with tumors lacking this cytokine production. In contrast to Kleine et al.,12 who reported a gradual disappearance of MCP-1 expression in normal cervical tissue via CIN towards cervical carcinoma tissue, we observed that all carcinoma cell lines expressed MCP-1 mRNA. In contrast, only one normal cervical epithelium expressed MCP-1 mRNA. Our results suggest that although the functional activity of antigen presenting cells (e.g. Langerhans’ cells and monocytes) seems to be depressed, antigen presenting cells are recruited to the tumor site. Macrophages have been reported to produce IL-6 and IL-6 has been reported to be a growth factor for cervical carcinoma cells.50-52 Thus tion of MCP-1, via chemoattraction of monocytes/macrophages and IL-6 produc-tion, may support tumor growth.
In conclusion, we have shown that after malignant transformation cervical epithelial cells have a decreased ability to express TNF-α, GM-CSF, IL-5, IL-10 and RANTES mRNA and an increased ability to express MCP-1 mRNA. (Loss of) expression of these cytokines by cervical cancer cells may modulate the local tumor environment thus supporting tumor growth.
A
C K N O W L E D G E M E N T SR
E F E R E N C E S1. IARC Working Group. IARC monographs on the evaluation of carcinogenic risk to humans. 1995.
2. Zur Hausen H. Papillomavirus infections--a major cause of human cancers. Biochim Biophys Acta 1996;1288(2): F55-78.
3. Wu T-C. Immunology of the human papilloma virus in relation to cancer. Curr Opin Immunol 1994;6:746-754.
4. Benton C, Shahidullah H, Hunter JA. Human papillomavirus in the immunosuppressed. Papillomavirus rep 1992;3:23-26.
5. Petry K, Scheffel D, Bode U, Gabrysiak T, Kochel H, Kupsch e, Blaubitz M, Niesert S, Kuhnle H, Schedel I. Cellular immunodeficiency enhances the progression of human papillomavirus-associ-ated cervical lesions. Int J Cancer 1994;57:836-840.
6. Ozsaran AA, Ates T, Dikmen Y, Zeytinoglu A, Terek C, Erhan Y, Ozcar, Tilgic A. Evaluation of the risk of cervical intraepithelial neoplasia and human papilloma virus infection in renal transplant patients receiving immunosuppressive therapy. Eur J Gynaecol Oncol 1999;20: 127-130. 7. Nakagomi H, Pisa P, Pisa E.K., Yamamoto Y, Halapi E, Backlin K. Lack of interleukin-2 (IL-2)
expression and selective expression of IL-10 mRNA in human renal cell carcinoma. Int J Cancer 1995;63:366-371.
8. Huang M, Wang J, Lee P, Meissner H. Human non-small cell lung cancer cells express a type 2 cytokine pattern. Cancer Res 1995;55:3847-3853.
9. Pao CC, Lin CY, Yao DS, Tseng CJ. Differential expression of cytokine genes in cervical cancer tissues. Biochem Biophys Res Commun 1995;214:1146-1151.
10. Tartour E, Gey A, Sastre Garau X, Lombard Surin I, Mosseri V, Fridman WH. Prognostic value of intratumoral interferon gamma messenger RNA expression in invasive cervical carcinomas. J Natl Cancer Inst 1998;90(4):287-294.
11. de Gruijl TD, Bontkes HJ, van den Muysenberg AJC, Meijer C, Walboomers J. Differences in cytokine mRNA profiles between premalignant and malignant lesions of the uterine cervix. Eur J Cancer 1999;35(3):490-497.
12. Kleine-Lowinski K, Gillitzer R, Kunhe-Heid R, Rosl F. Monocyte-Chemo-attractant-Protein-1 (MCP-1)-gene expression in cervical intra-epithelial neoplasias and cervical carcinomas. Int J Cancer 1999;82(1):6-11.
13. Woodworth CD, Simpson S. Comparative lymphokine secretion by cultured normal human cervical keratinocytes, papillomavirus-immortalized, and carcinoma cell lines. Am J Pathol 1993;142(5):1544-1555.
14. Linnell F, Mansson B. The prognostic value of eosinophilic leukocytes in the stroma of carci-noma of the cervix uteri: A study of 291 cases treated with radium and rontgen therapy. Acta Radiologica 1954;41:453-456.
15. Lasersohn JT, Thomas LB, Smith RR, Ketcham AS, Dillon JS. Carcinoma of the uterine cervix. Cancer 1964;17:338-351.
16. Bostrom SG, Hart WR. Carcinomas of the cervix with intense stromal eosinophilia. Cancer 1981;47:2887-2893.
17. Driel WJv, Hogendoorn PCW, Jansen FW, Zwinderman AH, Trimbos JB, Fleuren GJ. Tumor-associ-ated eosinophilic infiltrate of cervical cancer is indicative for a less effective immune response. Human Pathology 1996;27:904-911.
18. De Visser KE, Kast WM. Effects of TGF-β on the immune system: implications for cancer immu-notherapy. Leukemia 1999;13(8):1188-1199.
19. Clerici M, Merola M, Ferrario E, Trabattoni D, Villa ML, Stefanon B, Venzon DJ, Shearer GM, De PaloG, Clerici E. Cytokine production patterns in cervical intraepithelial neoplasia: association with human papillomavirus infection. J Natl Cancer Inst 1997;89(3):245-250.
20. van Driel WJ, Ressing ME, Brandt RM, Toes RE, Fleuren GJ, Trimbos JB, Kast WM, Melief CJ. The current status of therapeutic HPV vaccine. Ann Med 1996;28(6):471-477.
22. Koopman LA, Mulder A, Corver WE, Anholts JDH, Giphart MJ, Claas FHJ, Fleuren GJ. HLA class I phenotype and genotype alterations in cervical carcinomas and derivative cell lines. Tissue Antigens 1998;51:623-636.
23. Kaashoek JG, Mout R, Falkenburg JH, Willemze R, Fibbe WE, Landegent JE. Cytokine production by the bladder carcinoma cell line 5637: rapid analysis of mRNA expression levels using a cDNA-PCR procedure. Lymphokine Cytokine Res 1991;10(3):231-235.
24. Hein H, Schluter C, Kulke R, Christophers E, Schroder JM, Bartels J. Genomic organization, sequence analysis and transcriptional regulation of the human MCP-4 chemokine gene (SCYA13) in dermal fibroblasts: a comparison to other eosinophilic beta-chemokines. Biochem Biophys Res Commun 1999;255(2):470-476.
25. Meazza R, Verdiani S, Biassoni R, Coppolecchia M, Gaggero A, Orengo AM, Colombo MP, Azzarone B, Ferrini S. Identification of a novel interleukin-15 (IL-15) transcript isoform generated by alter-native splicing in human small cell lung cancer cell lines. Oncogene 1996;12:2187-2192. 26. Lim KG, Wan H, Bozza PT, Resnick MB, Wong DTW, Cruikshank WW, Kornfeld H, Center DM,
Weller PF. Human Eosinophils Elaborate the Lymphocyte Chemoattractants IL-16 (Lymphocyte Chemoattractant Factor) and RANTES. J Immunology 1996;156(7):2566-2570.
27. Hayes IM, Jordan NJ, Towers S, Smith G, Paterson JR, Earnshaw JJ, Roach AG, Westwick J, Williams RJ. Human vascular smooth muscle cells express receptors for CC chemokines. Arterioscler Thromb Vasc Biol 1998;18(3):397-403.
28. Bartels J, Schluter C, Richter E, Noso N, Kulke R, Christophers E, Schroder JM. Human dermal fibroblasts express eotaxin: molecular cloning, mRNA expression, and identification of eotaxin sequence variants. Biochem Biophys Res Commun 1996;225(3):1045-1051.
29. de Boer WI, van Schadewijk A, Sont JK, Sharma HS, Stolk J, Hiemstra PS, van Krieken JH. Trans-forming growth factor beta 1 and recruitment of macrophages and mast cells in airways in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1998;158(6):1951-1957.
30. Al Saleh W, Giannini SL, Jacobs N, Moutschen M, Doyen J, Boniver J, Delvenne P. Correlation of T-helper secretory differentiation and types of antigen-presenting cells in squamous intraepithe-lial lesions of the uterine cervix. J Pathol 1998;184(3):283-290.
31. Kunzendorf U, Hien Tran T, Bulfone-Paus S. The Th1-Th2 paradigm in 1998: law of nature or rule with exceptions. Nephrology Dialysis Transplantation 1998;13:2445-2448.
32. Alexandrow MG, Moses HL. Transforming growth factor beta and cell cycle regulation. Cancer Res 1995;55:1452-1457.
33. Chouaib S, Asselin Paturel C, Mami Chouaib F, Caignard A, Blay JY. The host-tumor immune con-flict: from immunosuppression to resistance and destruction. Immunol Today 1997:18(10);493-497.
34. Evander M, Frazer IH, Payne E, Qi YM, Hengst K, McMillan NAJ. Identification of the alpha6 integrin as a candidate receptor for papillomaviruses. J Virol 1997;71:2449-2456.
35. Frazer IH, Thomas R, Zhou Jeal. Potential strategies utilised by papillomavirus to evade host im-munity. Immunol Rev 1999;168:131-142.
36. Maeda H, Kuwahara H, Ichimura Y, Ohtsuki M, Kurakata S, Shiraishi A. TGF-beta enhances macro-phage ability to produce IL-10 in normal and tumor-bearing mice. J Immunology 1995;155:4926-4932.
37. Roberts AB, Sporn MB. Regulation of endothelial cell growth, architecture and matrix synthesis by TGF-beta. Am Rev Resp Dis 1989;140:1126-1128.
38. Koopman LA, Corver WE, Van der Slik AR, Giphart MJ, Fleuren GJ. Multiple genetic altera-tions cause frequent and heterogeneous HLA class I antigen loss in cervical cancer. J Ex Med 2000;191(6):961-976.
39. De Geest K, Bergman CA, Turyk ME, Frank BS, Wilbanks GD. Differential response of cervical intraepithelial and cervical carcinoma cell lines to transforming growth factor-beta 1. Gynecol Oncol 1994;55:376-385.
40. Kang SH, Won K, Chung H, Jong H, Song Y, Kim S, Bang YJ, Kim NK. Genetic integrity of trans-forming growth factor beta receptors in cervical carcinoma cell lines: loss of growth sensitivity but conserved transcriptional response to TGF-beta. Int J Cancer 1998;77:620-625.
acid and transforming growth factor-beta at late stages of human papillomavirus type 16-initiated transformation of human keratinocytes. Adv Exp Med Biol 1995;375:117-135.
42. Chen T, de Vries E, Hollema H, Yegen H, Vellucci V, Strickler H, Hildesheim A, Reiss M. Structural alterations of transforming growth factor-beta receptor genes in human cervical carcinoma. Int J Cancer 1999;82(1):43-51.
43. Chu T, Lai J, Shen CY, Liu H, Chao C. Frequent aberration of the transforming growth factor-beta receptor II gene in cell lines but no apparent mutation in pre-invasive and invasive carcinomas of the uterine cervix. Int J Cancer 1999;80(4): 506-510.
44. Santin AD, Hermonat PL, Hiserodt JC, Fruehauf J, Schranz V, Barclay D, Pecorelli S, Parham GP. Differential transforming growth factor-beta secretion in adenocarcinoma and squamous cell car-cinoma of the uterine cervix. Gynecol Oncol 1997;64(3):477-480.
45. Cumberbatch M, Kimber I. Tumour necrosis factor-alpha is required for accumulation of dendritic cells in draining lymph nodes and for optimal sensitization. Imm Today 1995;84:31-35.
46. Sugarman BJ, Aggarwal BB, Hass PE, Figari IS, Pallidino MAJr, Shephard HM. Recombinant human tumor necrosis factor alpha effects on proliferation of normal and transformed cells in vitro. Science 1985;230:943-945.
47. Peters JH, Gieseler R, Thiele B, Steinbach F. Dendritic cells: from ontogenetic orphans to myelo-monocytic descendants. Imm Today 1996;17:273-278.
48. Austyn JM. Dendritic cells. Curr Opin Hemat 1998;5:3-15.
49. Golumbek P, Azhari R, Jaffee EM, Levitsky HI, Lazenby A, Leong K, Pardol D. Controlled release biodegradable cytokine depots: a new approach in cancer vaccine design. Cancer Res 1993;53:5841-5844.
50. Tartour E, Gey A, Sastre Garau X, Pannetier C, Mosseri V, Kourilsky P, Fridman WH. Analysis of interleukin 6 gene expression in cervical neoplasia using a quantitative polymerase chain reaction assay: evidence for enhanced interleukin 6 gene expression in invasive carcinoma. Cancer Res 1994;54(23):6243-6248.
51. Pages F, Vives V, Sautes-Fridman C, Fossiez F, Berger A, Cugnenc PH, Tartour E, Fridman WH. Control of tumor development by intratumoral cytokines. Imm Lett 1999;68: 135-139.
