Indole-3-carbinol and diindolylmethane induce apoptosis of human cervical cancer cells and in murine HPV16-transgenic preneoplastic cervical epithelium.

Dietary indole-3-carbinol (I3C) has clinical benefits for both cervical cancer and laryngeal papillomatosis, and causes apoptosis of breast cancer cells in vitro. We asked whether I3C and its major acid-catalyzed condensation product diindolylmethane (DIM), which is produced in the stomach after consumption of cruciferous vegetables, could induce apoptosis of cervical cancer cell lines. We also asked whether this effect could be observed in vivo. In vitro, both I3C and DIM caused accumulation of DNA strand breaks in three cervical cancer cell lines. Induction of apoptosis was confirmed by nuclear morphology, nucleosome leakage, altered cytoplasmic membrane permeability and caspase 3 activation. Neither I3C nor DIM caused apoptotic changes in normal human keratinocytes. In C33A cervical cancer cells, DIM was more potent than I3C [dose at which the number of viable cells was 50% of that in untreated cultures (LD(50)) = 50-60 micromol/L for DIM and 200 micromol/L for I3C in a mitochondrial function assay] and faster acting. Furthermore, I3C reduced Bcl-2 protein in a time- and dose-dependent manner. In HPV16-transgenic mice, which develop cervical cancer after chronic estradiol exposure, apoptotic cells were detected in cervical epithelium by TdT-mediated dUTP nick-end labeling staining and by immunohistochemical staining of active caspase 3 only in mice exposed to 17beta-estradiol (E2) and fed I3C. Rare apoptotic cells were also observed by hematoxylin and eosin staining in the spinous layer of the cervical epithelium in both control and transgenic mice. Estradiol reduced the percentage of these late-stage apoptotic cells in the cervical epithelium of transgenic, E2-treated mice, but this reduction was prevented by I3C. These data confirm the proapoptotic action of I3C on transformed cells in vitro, extend the observations to cervical cancer cells and to DIM and show for the first time that dietary I3C results in increased apoptosis in target tissues in vivo.

Treatment of human papillomavirus gynecologic infections.

The future holds promise for better prevention and treatment of HPV infections. Currently, therapeutic options for HPV infections are limited, expensive, and often ineffective. Recent advances in understanding the basic virology and natural history of HPV infection have identified hitherto unsuspected approaches to therapy and have suggested novel pharmacologic and other types of interventions. An important area for future research is the need for detailed information about the mechanisms that trigger latent HPV and induce wart formation or promote malignant transformation. Conversely, we also need to know more about mechanisms that keep the virus in check (latent) in many HPV-infected persons.

Indole-3-carbinol prevents cervical cancer in human papilloma virus type 16 (HPV16) transgenic mice.

Mice that express transgenes for human papillomavirus type 16 under a keratin 14 promoter (K14-HPV16 mice) develop cervical cancer when they are given 17beta-estradiol chronically. We asked whether the antiestrogenic phytochemical indole-3-carbinol (I3C), found in cruciferous vegetables, administered at physiological doses, would prevent the cervical-vaginal cancer that is promoted in these mice by high doses of estrogen. We compared mice that were fed a control diet with those that were fed a diet supplemented with 2000 ppm I3C. In the group fed the control diet, at a dose of estradiol of 0.125 mg per 60-day release, 19 of 25 transgenic mice developed cervical-vaginal cancer within 6 months, and the remainder had dysplasia. Only 2 mice of 24 in the group fed the I3C supplemented diet developed cancer, and the remainder had dysplasia or hyperplasia. I3C reduced dysplasia in the nontransgenic mice. Similar results were obtained at a higher dose of estradiol (0.250 mg per 60-day release), and I3C helped to prevent morbidity associated with retention of fluid in the bladder that frequently occurred with the higher estradiol dose. Additionally, I3C appeared to reduce skin cancer in transgenic mice. These data indicate that I3C is a useful preventive for cervical-vaginal cancer and, possibly, other cancers with a papillomavirus component.

BRCA1 inhibition of estrogen receptor signaling in transfected cells.

Mutations of the breast cancer susceptibility gene BRCA1 confer increased risk for breast, ovarian, and prostatic cancers, but it is not clear why the mutations are associated with these particular tumor types. In transient transfection assays, BRCA1 was found to inhibit signaling by the ligand-activated estrogen receptor (ER-alpha) through the estrogen-responsive enhancer element and to block the transcriptional activation function AF-2 of ER-alpha. These results raise the possibility that wild-type BRCA1 suppresses estrogen-dependent transcriptional pathways related to mammary epithelial cell proliferation and that loss of this ability contributes to tumorigenesis.

Kinetics of HPV11 DNA replication after infection of keratinocytes with virions.

Human papillomavirus type 11 (HPV11) normally infects keratinocytes of stratified epithelia and replicates as an episome. To untangle early events in the development of a papilloma, we have infected human keratinocytes with human papillomavirus type 11 virions and monitored replication using density labeling with bromodeoxyuridine and subsequent density centrifugation. We show that only a portion of the virus reaching the nucleus undergoes replication and that continued replication occurs without recruitment from the nonreplicating pool of virus suggesting that HPV11 replication remains confined to a small number of cells. Increasing inoculum size leads to more viral DNA reaching the nucleus but not a corresponding increase in viral replication. Subsequent papilloma development must, therefore, occur within a small subset of cells.

Human papillomavirus type 11 transcripts are present at low abundance in latently infected respiratory tissues.

Respiratory tract tissues containing latent human papillomavirus (HPV) 11 were analyzed by reverse transcription-polymerase chain reaction for the presence of viral-specific RNA from the early region of the genome and compared to a similar analysis of laryngeal papillomas. Latently infected tissue contained low-abundance transcripts that could code for E1 and E2 proteins, but lacked evidence of spliced transcripts for the E6 and E7 proteins. Both latently infected tissue and papilloma tissue contained low-abundance antisense transcripts. Cultured cells infected with HPV 11 virions or transfected with HPV DNA, and cells derived from latently infected tissue, expressed transcripts similar to those seen in papillomas, but at a lower abundance. We postulate that latency is determined by the absence of or limiting levels of critical viral proteins.

Replicative intermediates of human papillomavirus type 11 in laryngeal papillomas: site of replication initiation and direction of replication.

We have examined the structures of replication intermediates from the human papillomavirus type 11 genome in DNA extracted from papilloma lesions (laryngeal papillomas). The sites of replication initiation and termination utilized in vivo were mapped by using neutral/neutral and neutral/alkaline two-dimensional agarose gel electrophoresis methods. Initiation of replication was detected in or very close to the upstream regulatory region (URR; the noncoding, regulatory sequences upstream of the open reading frames in the papillomavirus genome). We also show that replication forks proceed bidirectionally from the origin and converge 180 degrees opposite the URR. These results demonstrate the feasibility of analysis of replication of viral genomes directly from infected tissue.

Papillomaviruses in head and neck disease: pathophysiology and possible regulation.

Human papillomaviruses (HPVs) are etiologic agents of both benign and malignant epithelial tumors. More than 60 different types of viruses are known, each associated with tissue site and lesion type specificities and differing probabilities of malignant progression. HPVs type 6 and type 11 cause benign papillomas of mucosal squamous epithelium in the aerodigestive tract, with only rare conversion to malignancy. HPV 16 is the most frequently detected HPV in the genital tract, inducing flat lesions with a significant risk of malignant conversion. In the aerodigestive tract, HPV 16 is found only rarely in benign lesions but is detected in 5-20% of squamous carcinomas. In the aerodigestive tract, HPVs frequently cause latent infection, i.e., viral DNA present in tissue but no evidence of clinical or histologic disease. Approximately 10% of the general population may have latent infections. Regulation and activation of latent infections are not well understood, although it is clear that viral functions are tightly regulated by the state of differentiation of the squamous host cell. Control of viral transcription may be the key to prevention of viral activation, and thus control of disease. Among the possible agents under investigation are retinoids, growth factors, anti-sense RNA which interferes with viral expression, and estrogen metabolites. All of these agents modulate either viral expression or cell differentiation or both. It is hoped that in the near future one or more of these agents will be useful in preventing HPV-associated disease.

The interaction between HPV infection and estrogen metabolism in cervical carcinogenesis.

Cancer of the genital tract is the final outcome of some infections with human papillomavirus (HPVs), and the most estrogen-sensitive cells are at greatest risk for the HPV-related cancers. Therefore we investigated relationships between HPVs and estrogen metabolism in cells of the genital tract. Increased conversion of estradiol to 16 alpha-hydroxyestrone, known to be a risk factor for cancer in some other estrogen-sensitive cells, was investigated in keratinocytes from the genital tract. Primary cells, particularly those explants from the transformation zone of the cervix, are able to 16 alpha-hydroxylate estradiol. Both cervical and foreskin cells immortalized with HPV-16 are greatly enhanced in the 16 alpha-hydroxylation of estradiol as compared with normal cells. We suggest a model whereby the combined action of 16 alpha-hydroxylation of estrogen and HPV work together to promote cell proliferation.

A key DNA-protein interaction determines the function of the 5'URR enhancer in human papillomavirus type 11.

The 5' end of the upstream regulatory region (URR) of human papillomavirus type 11 (HPV-11) has enhancer function and binds cellular proteins. We have determined that one particular motif, a CCNGTNAC pair, is both necessary and sufficient for enhancer activity. This enhancement of expression can be competed in vivo by concatenated double-stranded oligonucleotides, indicating that protein-DNA binding is a requisite for enhancer activity. A 41-kDa protein, present in all epithelial cells assayed, binds to this enhancer motif. The 5'URR fragment functions as an enhancer both in primary keratinocytes from a variety of body sites and in fibroblasts. We conclude that tissue specificity is not a feature of this enhancer, and that the 41-kDa binding protein is ubiquitous. These data provide evidence that the 5'URR enhancer activity is dependent on only a few sequences and perhaps only one protein.

Identification of DNA-protein interactions and enhancer activity at the 5' end of the upstream regulatory region in human papillomavirus type 11.

We have examined the 5' end of the noncoding region of the genome of a human papillomavirus, HPV-11, for regulatory elements using permissive host cells. This region of unknown function in the upstream regulatory region (URR) is known to have unusual DNA structure and frequently contains rearrangements which are associated with some more virulent isolates. This 5' 269-bp fragment was found to exhibit both specific DNA-protein binding using laryngeal papilloma protein extracts and enhancer activity in normal and papillomatous primary laryngeal cells. The viral DNA flanks the L1 open reading frame and does not contain the viral E2 binding site. Three distinct protein binding sites are contained in a 50-bp region of the fragment. This fragment, as a whole, functions as an enhancer in primary laryngeal and papilloma cells when ligated to the SV40 promoter and SV40 T-antigen gene. We conclude that this part of the noncoding region of the papillomaviruses has elements characteristic of regulatory elements in cells permissive for infection by these viruses.

Tissue site-specific enhancer function of the upstream regulatory region of human papillomavirus type 11 in cultured keratinocytes.

The human papillomavirus type 11 regulatory region was cloned upstream of a reporter complex and microinjected into nuclei of individual primary human keratinocytes. Genital and laryngeal keratinocytes, normal host cells for this virus, exhibited higher levels of expression than cutaneous keratinocytes. We conclude that a papillomavirus enhancer(s) shows preferences among epithelial cells from different tissue sites.

Simian virus 40 (SV40) T antigen binds specifically to double-stranded DNA but not to single-stranded DNA or DNA/RNA hybrids containing the SV40 regulatory sequences.

Simian virus 40 T antigen has been shown previously to bind specifically with high affinity to sites within the regulatory region of double-stranded simian virus 40 DNA. Using competition filter binding and the DNA-binding immunoassay, we show that T antigen did not bind specifically to either early or late single-stranded DNA containing these binding sites. Moreover, T antigen did not bind these sequences present in single-stranded RNA, RNA/RNA duplexes, or RNA/DNA hybrids. T antigen did, however, bind as efficiently to single-stranded DNA-cellulose as to double-stranded DNA-cellulose. This binding was nonspecific because it was independent of the presence of T-antigen-binding sites. The implications of these observations are discussed.

Adenovirus type 2 DNA replicated in arginine-starved KB cells is infectious.

Adenovirus type 2 DNA from extracts of KB cells rendered nonpermissive by arginine starvation is infectious.