p16/p14(ARF) cell cycle regulatory pathways in primary neuroblastoma: p16 expression is associated with advanced stage disease.

p16 regulates the G(1)-S cell cycle transition by inhibiting the cyclin D-cyclin-dependent kinase (CDK)4/CDK6-mediated phosphorylation of retinoblastoma protein (pRb). We examined the possible derangement of the p16-CDK/cyclin D-pRb pathway in 40 primary neuroblastomas including 18 samples in the unfavorable stages (C and D) and 22 in the favorable stages (A, B, and Ds) by PCR, reverse transcription-PCR, Western blot, and immunohistochemistry and correlated the results with clinical outcome. No samples harbored alterations of the p16 gene. Interestingly, the samples in the unfavorable stages exhibited expression of p16 mRNA and protein more frequently than those in the favorable stages [mRNA, 9 of 18 (50%) versus 2 of 22 (9%), P = 0.006; protein, 5 of 16 (31%) versus 0 of 18 (0%), P = 0.013]. Alterations of the downstream components of the pathway were infrequent. pRb was deregulated in the majority of samples investigated [27 of 33 (82%), 24 with hyperphosphorylated pRb and 3 with no pRb protein]. The phosphorylation status of pRb did not correlate with p16 protein expression, suggesting that the elevated p16 protein may not be functioning properly to regulate the pathway. Among patients of all stages, p16 expression was significantly associated with a lower overall survival. There was no overexpression of MDM2, and loss of p14(ARF) expression and p53 mutation were infrequent events. Taken together, these findings suggest that up-regulated p16 expression may represent a unique feature of aggressive neuroblastoma.

In vitro sensitivity of T-cell lymphoblastic leukemia to UCN-01 (7-hydroxystaurosporine) is dependent on p16 protein status: a Pediatric Oncology Group study.

p16 regulates the cell cycle pathway by inhibiting the cyclin Ds-cyclin-dependent kinase (CDK) 4/6-mediated phosphorylation of retinoblastoma protein (pRb). Previously, we reported that most primary T-cell acute lymphoblastic leukemia (T-ALL) harbored p16 inactivation and hyperphosphorylated pRb without cyclin Ds or CDK4/6 alterations. Therefore, inhibiting CDK4/6 may be an ideal therapeutic approach for p16 (-) T-ALL. UCN-01 (7-hydroxystaurosporine) is a potent antitumor agent that exerts its effects through the inhibition of CDKs. We now report that p16 protein expression status of T-ALL cells influences their sensitivity to UCN-01. In 36 primary T-ALL cells, the IC50s of UCN-01 in the 27 p16 (-) cells (43+/-52 nM) was significantly lower than that in the 9 p16 (+) cells (258+/-260 nM). Our results suggest that agents like UCN-01 may be useful as a p16-selective therapy for T-ALL.

Retinoic acid resistance at late stages of human papillomavirus type 16-mediated transformation of human keratinocytes arises despite intact retinoid signaling and is due to a loss of sensitivity to transforming growth factor-beta.

In our in vitro model of human cell carcinogenesis, normal human foreskin keratinocytes (HKc) transfected with human papillomavirus type 16 DNA (HKc/HPV16) progress toward malignancy through several phenotypically defined and reproducible "steps" that include immortalization, growth factor independence (HKc/GFI), differentiation resistance (HKc/DR), and ultimately malignant conversion. While HKc/HPV16 are very sensitive to growth inhibition by all-trans-retinoic acid (RA) at early passages, they lose their sensitivity to RA during progression in culture. However, gel mobility shift assays using the retinoid response elements DR1 and DR5 showed no changes in binding activity of nuclear extracts obtained from HKc/HPV16 at different stages of in vitro progression. Similarly, Western blot analyses for retinoic acid receptor gamma-1 and the retinoid X receptors failed to reveal any decreases in the levels of these retinoid receptors throughout progression. In addition, luciferase activity driven by the SV40 promoter with a DR5 enhancer element was activated following RA treatment of HKc/DR that were resistant to growth inhibition by RA. Since RA induces transforming growth factor-beta2 (TGF-beta2) in normal HKc and HKc/HPV16, we investigated whether this response changed during progression. Again, RA induced TGF-beta2 mRNA in early and late passage HKc/HPV16, HKc/GFI, and HKc/DR approximately to the same extent, confirming that the RA signaling pathways remained intact during in vitro progression despite the fact that the cells become resistant to growth inhibition by RA. We then investigated the sensitivity of HKc/HPV16 to growth inhibition by TGF-beta. While early passage HKc/HPV16 were as sensitive as normal HKc to growth inhibition by TGF-beta1 and TGF-beta2, the cells became increasingly resistant to both TGF-beta isotypes during in vitro progression. In addition, while both RA and TGF-beta produced a decrease in the levels of mRNA for the HPV16 oncogenes E6 and E7 in early passage HKc/HPV16, this effect was also lost at later stages of progression. Finally, blocking anti-TGF-beta antibodies partially prevented RA inhibition of growth and E6/E7 expression in early passage HKc/HPV16. Taken together, these data strongly suggest that inhibition of growth and HPV16 early gene expression in HKc/HPV16 by RA is mediated by TGF-beta and that a loss of RA sensitivity is linked to TGF-beta resistance rather than alterations in RA signaling.

Universal inactivation of both p16 and p15 but not downstream components is an essential event in the pathogenesis of T-cell acute lymphoblastic leukemia.

p16/p15 regulate the cell cycle pathway by inhibiting the cyclin Ds-CDK4/6 mediated phosphorylation of pRb. We reported previously that in T-cell acute lymphoblastic leukemia (T-ALL), p16 and p15 were frequently (approximately 70%) inactivated at the DNA level by deletion, mutation, or hypermethylation. Therefore, we hypothesize that inactivation of the cell cycle regulatory pathway may be essential in the pathogenesis of T-ALL, and that the remaining T-ALL with a wild-type p16/p15 gene likely harbor inactivation of these genes at RNA or protein levels. Alternatively, the downstream components of the pathway including CDK4/6, cyclin Ds, and pRb may be deregulated. In 124 primary T-ALLs, we found inactivation of the p16 and p15 genes at the DNA level in 79 (64%) and 64 (52%) samples, respectively. Only 9 of the 45 samples with wild-type p16 expressed p16 protein, whereas the remaining 36 lacked p16 expression at the RNA or protein level. In the 60 samples with an intact p15 gene, only 2 expressed p15 mRNA, and the only one analyzed lacked p15 protein. Overall, the abrogation rates for p16 and p15 at DNA/RNA/protein levels were 93% (115 of 124) and 99% (123 of 124), respectively. Although no alterations were evident in cyclin Ds or CDK4/6, pRb was hyperphosphorylated in the majority of samples investigated. These findings strongly support that both p16 and p15 are specific targets in the deregulation of the cell cycle pathway in T-ALL and that the inactivation of these genes is most likely essential in the pathogenesis of this disease.

Methylation of p16INK4A in primary gynecologic malignancy.

The p16INK4A gene mapped on band p21 of chromosome 9 can be inactivated via multiple mechanisms including homozygous deletion, point mutation and promoter hypermethylation in various human tumors. A polymerase chain reaction (PCR) based analysis was performed to examine methylation of the p16INK4A gene promoter in 196 primary gynecologic malignancies including 98 cervical, 49 endometrial and 49 ovarian carcinomas. Methylation of p16INK4A was detected in 31% of cervical, 20% of endometrial, and 4% of ovarian carcinomas, respectively. The incidence of p16INK4A methylation in patients with cervical and endometrial carcinomas at advanced stages (stages III-IV) was statistically higher than those at early stages (stages I-II). There were also significant differences in the incidence of p16INK4A methylation in both cancers between the patients who had died of their disease or were alive with evidence of disease, and those without evidence of disease. The results indicate that methylation of the p16INK4A gene is present in a proportion of primary gynecologic malignancies and this alteration may be associated with poor outcome in cervical and endometrial carcinomas.

Use of alanosine as a methylthioadenosine phosphorylase-selective therapy for T-cell acute lymphoblastic leukemia in vitro.

Methylthioadenosine phosphorylase (MTAP) is an important enzyme for the salvage of adenine and methionine and is deficient in a variety of cancers including T-cell acute lymphocytic leukemia (T-ALL). Previously, we reported that the MTAP gene was deleted in over 30% of T-ALL patients at both diagnosis and relapse. We now report that MTAP-primary T-ALL cells are more sensitive to the toxicity of L-alanosine, an inhibitor of de novo AMP synthesis, than are MTAP+ primary T-ALL cells. As measured by [3H]thymidine incorporation, DNA synthesis in all seven MTAP-primary T-ALL cells was inhibited by L-alanosine with a mean IC50 of 4.8+/-5.3 ILM (range, 0.3-11.3 microM). On the other hand, the IC50 for 60% (12 of 20) of MTAP+ primary T-ALL was 19+/-18 microM (range, 1.7-67 microM; P = 0.02), whereas the remaining 40% (8 of 20) had an IC50 of >80 microM4. Furthermore, normal lymphocytes and MTAP+ primary T-ALL cells were rescued from L-alanosine toxicity by the MTAP substrate 5'-deoxyadenosine, but MTAP-T-ALL cells were not. These results indicate that normal cells, which are intrinsically MTAP+, would be protected from L.-alanosine toxicity, whereas MTAP-tumor cells would be killed. Thus, our results support the use of L-alanosine alone or in combination with a salvage agent as a MTAP-selective therapy and therefore lay the foundation for the initiation of clinical trials for the treatment of T-ALL and other MTAP-deficient malignancies with L-alanosine.

Frequent deregulation of p16 and the p16/G1 cell cycle-regulatory pathway in neuroblastoma.

Alterations of the p16 gene in neuroblastoma are very rare. Pronounced expression of p16 at both the transcript and protein levels, however, was observed in 7 of 19 (39%) neuroblastoma cell lines and 2 of 6 (33%) primary neuroblastoma samples. As p16 expression is tightly controlled in a feedback loop with Rb, we investigated the possibility that changes in p16 expression were reflective of alterations of the downstream components in the G1 regulatory pathway. Two cell lines and one primary sample highly expressing p16 were shown to harbor CDK4 amplification. The cyclin D2 gene was infrequently expressed in neuroblastoma cell lines and did not correlate with p16 expression. Slight variations in the expression of CDK6, cyclins D1, D3 and E; and E2F1 and E2F2 among the cell lines were observed, without apparent correlation with p16 status. No mutations to the p16-binding site of CDK4 and CDK6 nor any mutations to the coding region of p16 itself were identified in neuroblastoma cell lines. Despite frequent N-myc amplification in these cell lines, no relationship with this gene was observed either. All cell lines contained Rb protein with varying degrees of phosphorylation, which bears no correlation with p16 expression. Overall, alterations of the G1 pathway in neuroblastoma included relatively frequent p16 expression and infrequent CDK4 amplification and cyclin D2 expression. Despite a reported feedback relationship between p16 expression and Rb/G1 deregulation, p16 expression in neuroblastoma cell lines is independent of Rb gene and phosphorylation status and, in contrast to other cell lines where expression of p16 leads to G1/S arrest, neuroblastoma cell lines proliferate in the presence of elevated levels of p16.

The Ch14.18-GM-CSF fusion protein is effective at mediating antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity in vitro.

Granulocyte/macrophage-colony stimulating factor (GM-CSF) is very effective at enhancing antibody-dependent cellular cytotoxicity (ADCC) mediated by granulocytes and monocytes. Recently, a fusion protein consisting of GM-CSF and chimeric human/mouse anti-ganglioside G(D2) antibody Ch14.18 (Ch14.18-GM-CSF) has been generated to improve the effectiveness of immunotherapy by directing GM-CSF to the tumor microenvironment and prolonging its relatively short half-life. In this study, we examined the ability of this fusion protein to enhance the in vitro killing of G(D2)-expressing human neuroblastoma cells by granulocytes and mononuclear cells, as well as by complement. The Ch14.18-GM-CSF fusion protein was equally effective as the combination of equivalent amounts of free Ch14.18 and GM-CSF in mediating the killing of NMB7 neuroblastoma cells by granulocytes from seven of eight neuroblastoma patients. The fusion protein was also equally effective as the combination of Ch14.18 and GM-CSF in mediating ADCC by neuroblastoma patients' mononuclear cells. In addition, the fusion protein was as effective as Ch14.18 alone in directing complement-dependent cytotoxicity against NMB7 cells. Our results demonstrate that the biological activities expressed by ADCC and complement-dependent cytotoxicity of both monoclonal antibody Ch14.18 and GM-CSF are retained by the Ch14.18-GM-CSF fusion protein and lend further support for future clinical trials of this fusion protein in patients with neuroblastoma.

Shortened survival after relapse in T-cell acute lymphoblastic leukemia patients with p16/p15 deletions.

p16 Alterations were detected in > 60% of 103 primary T-ALL samples. In paired diagnosis-relapse patient samples, 80% of the relapse samples with p16 deletion were deleted at diagnosis. When p16 was homozygously deleted, p15 gene alterations were found in 72% of the diagnosis T-ALL patient samples, increasing significantly to 100% at relapse. Alterations of p18 were not detected. No clinical significance of p15/p16 gene deletion in diagnosis T-ALL was found with respect to white blood cell (WBC) count, incidence of mediastinal mass, rate of relapse, duration of first remission or event-free survival. In relapse T-ALL, however, patients with p16 deletion experienced a significantly shorter duration of post-relapse survival, demonstrating that p16 deletion is clinically significant in T-ALL.

Frequent and selective methylation of p15 and deletion of both p15 and p16 in T-cell acute lymphoblastic leukemia.

Frequent deletion of chromosome 9p21 in many cancers has suggested the presence of tumor suppressor genes in this region. Two genes mapping to 9p21, p15 and p16, encode inhibitors for cyclin-dependent kinases 4 and 6. We recently found that in T-cell acute lymphoblastic leukemia (T-ALL), both the p15 and p16 genes are deleted at a high frequency, with p16 gene deletion occurring slightly more frequently than p15 gene deletion. We now show that in addition to deletion, the p15 gene is preferentially hypermethylated at a 5' CpG island, which has been shown previously to be associated with loss of transcription of this gene. The p15 gene was methylated in 38% (17 of 45) of T-ALL patients at diagnosis and in 22% (7 of 32) of patients at relapse. On the other hand, methylation of the p16 gene was a rare event, occurring in 4% (2 of 49) of patients at diagnosis and in none (0 of 30) at relapse. The overall rates of alteration occurring in at least one allele of the p15 gene is 84% at diagnosis and 88% at relapse. These rates are as high as, if not greater than, those for the p16 gene (80% at diagnosis and 74% at relapse). In fact, such alterations involve both alleles in the majority of samples: 76% for p15 and 67% for p16 at diagnosis. All together, more than one-half (56%) of T-ALL samples harbor alterations in both alleles of both p15 and p16. These results lend strong support for a role of both p15 and p16 as tumor suppressors in T-ALL.

Presence of methylthioadenosine phosphorylase (MTAP) in hematopoietic stem/progenitor cells: its therapeutic implication for MTAP (-) malignancies.

Methylthioadenosine phosphorylase (MTAP) is important for the salvage of adenine and methionine. Recently, we found frequent deletion of MTAP in T-cell acute lymphoblastic leukemia (T-ALL) patients both at diagnosis and at relapse (A. Batova et al., Blood, 88: 3083-3090, 1996). In addition, MTAP deficiency has been reported in other cancers. Thus, MTAP deficiency in cancer may offer opportunities for developing selective therapy, which would spare normal cells. It is therefore important to document the presence of MTAP activity in hematopoietic stem/progenitor cells. Our approach was to investigate whether hematopoietic stem/progenitor cells can be rescued from the cytotoxicity of an AMP synthesis inhibitor, L-alanosine, by 5'-deoxyadenosine, a process that requires MTAP. Erythroid burst-forming unit, granulocyte/monocyte colony-forming unit, or granulocyte/erythrocyte/macrophage/megakaryocyte colony-forming unit progenitors and the primitive high proliferative potential colony-forming cells in the purified CD34(+) cells were cultured in horse serum-containing medium, and their colony growth was found to be suppressed by incubation with 5 microM or greater concentrations of L-alanosine. However, in the presence of 5-10 microM of 5'-deoxyadenosine, colony formation of hematopoietic stem/primitive progenitors was restored. On the other hand, 5'-deoxy-5'-methylthioadenosine, the endogenous substrate of MTAP, was toxic to hematopoietic stem/progenitors (ID50 < 1 microM), presumably due to inhibition of methylation reactions or polyamine synthesis. We also compared the effects of L-alanosine and 5'-deoxyadenosine on MTAP (+) and MTAP (-) T-ALL cell lines. Treatment of MTAP (+) Molt 4 and MTAP (-) CEM cell lines with L-alanosine in the presence of 5'-deoxyadenosine resulted in killing of MTAP (-), but not MTAP (+) cells. Therefore, our findings demonstrate the presence of MTAP in human hematopoietic stem/progenitor cells and support the possibility of targeting MTAP in the design of an enzyme-selective therapy for T-ALL and other MTAP-deficient malignancies.

Frequent deletion in the methylthioadenosine phosphorylase gene in T-cell acute lymphoblastic leukemia: strategies for enzyme-targeted therapy.

Methylthioadenosine phosphorylase (MTAP), an enzyme essential for the salvage of adenine and methionine, is deficient in a variety of cancers, including acute lymphoblastic leukemia (ALL). Because the MTAP gene is located adjacent to the tumor-suppressor gene p16 on chromosome 9p21 and more than 60% of T-cell ALL (T-ALL) patients have deletion in the p16 gene, we examined the status of the MTAP gene in T-ALL patients. Quantitative polymerase chain reaction amplification of exon 8 of MTAP showed a deletion in 16 of 48 (33.3%) patients at diagnosis and in 13 of 33 (39.4%) patients at relapse. Southern blot analysis showed that, in addition to deletion of the entire MTAP gene, a common break point was between exons 4 and 5, resulting in deletion of exons 5 through 8. The finding of frequent deficiency of MTAP in T-ALL offers the possibility of an enzyme targeted therapy for T-ALL. MTAP(-) T-ALL-derived cell line, CEM cells were very sensitive to methionine deprivation, with cell viability at 50% of control as early as 48 hours after methionine deprivation. In contrast, methionine deprivation had little effect on the viability of normal lymphocytes or on their proliferative response to phytohemagglutinin. Alanosine, an inhibitor of AMP synthesis, inhibited the growth of both MTAP(+) (Molt-4 and Molt-16) and MTAP(-) (CEM and HSB2) cell lines. However, the addition of methylthioadenosine, the substrate of MTAP, protected the MTAP(+) cells but not the MTAP(-) cells from alanosine toxicity. These findings suggest the possibility of targeting MTAP for selective therapy of T-ALL.

The p16 and p18 tumor suppressor genes in neuroblastoma: implications for drug resistance.

The cyclin dependent kinase inhibitors p16 and p18 were investigated in neuroblastoma. Only one of 19 neuroblastoma cell lines, an adriamycin-resistant variant, and none of 5 primary neuroblastoma, was deleted for p16 while its parental drug sensitive cell line is p16 intact. The region of deletion minimally extended centromeric to include p15, and telomeric to interferon-beta. This is the first report of a p16 gene alteration in neuroblastoma. No p16 gene hypermethylation or mutations were found. No homozygous deletions of p18 in these samples were found, although several instances of loss of heterozygosity are suspected. No p18 point mutations were detected. We conclude that (1) neither p16 nor p18 are likely involved in the pathogenesis of neuroblastoma; and (2) the role of p16, or another 9p21 gene, in the development of drug resistance warrants further investigation.

Genomic cloning of methylthioadenosine phosphorylase: a purine metabolic enzyme deficient in multiple different cancers.

5'-Deoxy-5'-methylthioadenosine phosphorylase (methylthioadeno-sine: ortho-phosphate methylthioribosyltransferase, EC 24.2.28; MTAP) plays a role in purine and polyamine metabolism and in the regulation of transmethylation reactions. MTAP is abundant in normal cells but is deficient in many cancers. Recently, the genes for the cyclin-dependent kinase inhibitors p16 and p15 have been localized to the short arm of human chromosome 9 at band p21, where MTAP and interferon alpha genes (IFNA) also map. Homozygous deletions of p16 and p15 are frequent malignant cell lines. However, the order of the MTAP, p16, p15, and IFNA genes on chromosome 9p is uncertain, and the molecular basis for MTAP deficiency in cancer is unknown. We have cloned the MTAP gene, and have constructed a topologic map of the 9p21 region using yeast artificial chromosome clones, pulse-field gel electrophoresis, and sequence-tagged-site PCR. The MTAP gene consists of eight exons and seven introns. Of 23 malignant cell lines deficient in MTAP protein, all but one had complete or partial deletions. Partial or total deletions of the MTAP gene were found in primary T-cell acute lymphoblastic leukemias (T-ALL). A deletion breakpoint of partial deletions found in cell lines and primary T-ALL was in intron 4. Starting from the centromeric end, the gene order on chromosome 9p2l is p15, p16, MTAP, IFNA, and interferon beta gene (IFNB). These results indicate that MTAP deficiency in cancer is primarily due to codeletion of the MTAP and p16 genes.

Progressive loss of sensitivity to growth control by retinoic acid and transforming growth factor-beta at late stages of human papillomavirus type 16-initiated transformation of human keratinocytes.

Retinoids (vitamin A and its natural and synthetic derivatives) have shown potential as chemopreventive agents, and diets poor in vitamin A and/or its precursor beta-carotene have been linked to an increased risk of cancer at several sites including the cervix. Human papillomavirus (HPV) plays an important role in the etiology of cervical cancer. We have developed an in vitro model of cancer progression using human keratinocytes (HKc) immortalized by HPV16 DNA (HKc/HPV16). Although immortal, early passage HKc/HPV16, like normal HKc, require epidermal growth factor (EGF) and bovine pituitary extract (BPE) for proliferation and undergo terminal differentiation in response to serum and calcium. However, following prolonged culture, growth factor independent HKc/HPV16 lines that no longer require EGF and BPE can be selected (HKc/GFI). Further selection of HKc/GFI produces lines that are resistant to serum- and calcium- induced terminal differentiation (HKc/DR). HKc/DR, but not early passage HKc/HPV16, are susceptible to malignant conversion following transfection with viral Harvey ras or Herpes simplex virus type II DNA. We have investigated the sensitivity of low to high passage HKc/HPV16 and HKc/GFI to growth control by all-trans-retinoic acid (RA, an active metabolite of vitamin A). Early passage HKc/HPV16 are very sensitive to growth inhibition by RA, and in these cells RA decreases the expression of the HPV16 oncogenes E6 and E7. However, as the cells progress in culture they lose their sensitivity to RA. Growth inhibition by RA may be mediated through the cytokine transforming growth factor-beta (TGF-beta), a potent inhibitor of epithelial cell proliferation. RA treatment of HKc/HPV16 and HKc/GFI results in a dose-and time-dependent induction (maximal of 3-fold) in secreted levels of TGF-beta. Also, Northern blot analysis of mRNA isolated from HKc/HPV16 demonstrated that RA treatment induced TGF-beta 1 and TGF-beta 2 expression about 3- and 50-fold, respectively. We next studied the effect of TGF-beta 1 and TGF-beta 2 on the proliferation of early to late passage HKc/HPVa6, HKc/GFI and HKc/DR. While early passage HKc/HPV16 were as sensitive as normal HKc to growth inhibition by TGF-beta 1 and TGF-beta 2, the cells became increasingly resistant to TGF-beta during in vitro progression, with the proliferation of HKc/DR being virtually unaffected by TGF-beta 1 or TGF-beta 2 treatment. Overall, loss of growth inhibition by RA parallels loss of TGF-beta sensitivity.(ABSTRACT TRUNCATED AT 400 WORDS)

Increased levels and constitutive tyrosine phosphorylation of the epidermal growth factor receptor contribute to autonomous growth of human papillomavirus type 16 immortalized human keratinocytes.

Transfection of individual normal human foreskin keratinocyte (HKc) strains with human papillomavirus type 16 (HPV16) DNA results in the establishment of immortalized cell lines (HKc/HPV16) which, like normal HKc, require epidermal growth factor (EGF) and bovine pituitary extract (BPE) for proliferation in serum-free media. However, sublines which proliferate in serum-free media in the absence of EGF and BPE can be reproducibly established from individual HKc/HPV16 lines, following selection in serum-free media lacking EGF and BPE. The growth factor-independent sublines (HKc/GFI) proliferate in the absence of EGF and BPE at the same rate and to the same extent as in medium supplemented with these growth factors, whereas the parental HKc/HPV16 lines proliferate poorly in the absence of EGF and BPE. As a first approach to understanding the molecular basis by which HKc/GFI have lost their requirement for EGF, we compared EGF uptake and EGF receptor (EGFR) numbers in normal HKc, HKc/HPV16, and HKc/GFI. HKc/GFI exhibit increased EGF uptake and increased EGFR numbers compared to HKc/HPV16. A neutralizing antibody against the extracellular domain of the EGFR dramatically inhibited clonal growth of HKc/GFI, indicating that signaling through the EGFR must be important for the ability of HKc/GFI to proliferate in the absence of EGF. In addition, while in the absence of EGF normal HKc and HKc/HPV16 exhibited no detectable EGFR tyrosine phosphorylation, the EGFRs in HKc/GFI were tyrosine phosphorylated in the absence of EGF and hyperphosphorylated in the presence of EGF. Although an anti-TGF-alpha antibody inhibited the growth of HKc/GFI, we unexpectedly found that HKc/GFI and HKc/HPV16 secreted comparable and extremely low amounts of TGF-alpha (4 to 9 pg/10(6) cells per 24 h); about 100- to 250-fold less than normal HKc (1018 pg/10(6) cells per 24 h). No other ligands for the EGFR were detected in media conditioned by normal HKc, HKc/HPV16, or HKc/GFI. Thus, while overexpression and constitutive activation of the EGFR appear to be important features of HKc/GFI, enhanced secretion of TGF-alpha or other ligands for the EGFR does not explain the proliferation of HKc/GFI in the absence of EGF and BPE.

Retinoic acid suppresses human papillomavirus type 16 (HPV16)-mediated transformation of human keratinocytes and inhibits the expression of the HPV16 oncogenes.

We have used a model system of normal HKc and HKc immortalized by transfection with HPV16 DNA (HKc/HPV16) to investigate the effect of RA on the growth of HKc/HPV16 and the expression of the HPV16 oncogenes E6 and E7. These studies found that HKc/HPV16 are about 100-fold more sensitive than normal HKc to growth inhibition by RA in both clonal and mass culture growth assays. The precursor to RA, retinol, was also found to be a more potent inhibitor of growth of HKc/HPV16 than normal HKc while beta-carotene did not inhibit growth of either normal HKc or HKc/HPV16. No differences were observed in the rate of uptake of [3H]RA or [3H]retinol between normal HKc and HKc/HPV16. Northern blot analysis of mRNA extracted from HKc/HPV16 cultured in the absence or in the presence of 10(-7) M RA showed that the expression of the HPV16 oncogenes E6 and E7 as well as the early ORFs E2 and E5 is substantially reduced following RA treatment. In addition, protein levels of E6 and E7, as measured by immunofluorescence (E6 and E7) and Western blot (E7) are also decreased by RA treatment of HKc/HPV16. Since E6 and E7 are considered the oncogenes of HPV16, we explored the possibility that RA may interfere with HPV16-mediated immortalization of HKc. The RA treatment (1 nM) of normal HKc, during or immediately following transfection with HPV16 DNA, inhibited immortalization by about 95%. Overall, these results provide a direct biochemical basis for a role of dietary retinoids in the chemoprevention of HPV-induced cancers.

Retinoic acid induces secretion of latent transforming growth factor beta 1 and beta 2 in normal and human papillomavirus type 16-immortalized human keratinocytes.

Similar cellular responses are elicited by retinoic acid (RA) and transforming growth factor beta (TGF-beta). We investigated the ability of RA to modulate the production of TGF-beta in normal human keratinocytes (HKc) and HKc lines immortalized by transfection with human papillomavirus type 16 DNA (HKc/HPV16). RA treatment of both normal HKc and HKc/HPV16 resulted in a 2-3-fold induction in secreted levels of latent TGF-beta. The induction in TGF-beta secretion by RA was dose dependent, with significant increases observed with RA concentrations as low as 1-10 nM, and time dependent, with maximal induction occurring about 3 days after initiation of RA exposure. In addition, RA induced intracellular levels of TGF-beta almost 5-fold. Sandwich enzyme-linked immunosorbent assays were used to specifically quantify TGF-beta 1 and TGF-beta 2 secreted by normal HKc and HKc/HPV16 cultured in the absence or presence of RA. RA increased the secreted levels of latent TGF-beta 1 and TGF-beta 2 an average of 2- and 5-fold, respectively, with no major differences in the fold induction between normal HKc and HKc/HPV16. Northern blot analysis of mRNA isolated from HKc/HPV16 demonstrated that RA treatment induced specific transcripts for TGF-beta 1 and TGF-beta 2 about 3- and 50-fold, respectively. RA treatment of HKc had no significant effect on the binding affinity of TGF-beta for its receptors or receptor number. Normal HKc and HKc/HPV16 displayed similar dose-dependent inhibition of proliferation by TGF-beta 1. These studies indicate that RA may regulate growth control in both normal HKc and HKc/HPV16 by enhancing TGF-beta 1 and TGF-beta 2 production, which, after activation at the cell surface, could inhibit cellular proliferation in an autocrine and/or paracrine manner.

Retinoic acid and calcitriol inhibition of growth and NADH oxidase of normal and immortalized human keratinocytes.

Plasma membranes were isolated by aqueous two-phase partition from normal human keratinocytes (HKc) and from human keratinocytes immortalized with human papillomavirus type 16 DNA (HKc/HPV16). The NADH oxidase of plasma membrane vesicles of normal HKc was stimulated by epidermal growth factor whereas that of HKc/HPV16 was not. The NADH oxidase of the plasma membranes from both normal HKc and HKc/HPV16 was inhibited by calcitriol (1 alpha-1,25-dihydroxy vitamin D-3) and retinoic acid. However, with plasma membranes from HKc/HPV16 the NADH oxidase was more susceptible to inhibition by retinoic acid than were membranes from normal HKc. Similarly, clonal growth of HKc/HPV16 was inhibited by retinoic acid at lower concentrations than normal HKc whereas inhibition of clonal growth of normal HKc and HKc/HPV16 by calcitriol showed similar dose-dependencies. Comparing normal HKc and HKc/HPV16, the results demonstrate parallel inhibition of clonal growth and NADH oxidase by both retinoic acid and calcitriol of HKc/HPV16 but not of normal HKc. These results suggest that an increased sensitivity of the plasma membrane NADH oxidase of HKc/HPV16 to retinoic acid may be related to the increased sensitivity of these cells to growth control by retinoic acid. In addition, since plasma membrane NADH oxidase of HKc/HPV16 shows altered responsiveness to growth modulators such as EGF, retinoic acid and calcitriol, it appears that HKc/HPV16 express an NADH oxidase with different characteristics than those of normal HKc.

Increased sensitivity of human keratinocytes immortalized by human papillomavirus type 16 DNA to growth control by retinoids.

Human papillomavirus (HPV) type 16 (HPV16) is associated with a large percentage of cervical malignancies, and HPV16 DNA can immortalize human keratinocytes in vitro. The transforming ability of the virus resides primarily in the open reading frames E6 and E7. Retinoids are potent modulators of growth and differentiation of keratinocytes and have been shown to reverse cervical lesions resulting from HPV infection. We compared the sensitivity of normal human foreskin keratinocytes (HKc) and four immortalized HKc lines, independently obtained by transfection of different normal HKc strains with HPV16 DNA (HKc/HPV16), to growth control by retinoic acid (RA). All the HKc/HPV16 lines were 10- to 100-fold more sensitive than normal HKc to growth inhibition by RA in both clonal and mass culture growth assays. The precursor to RA, retinol, was also found to be a more potent inhibitor of growth of HKc/HPV16 than normal HKc, while beta-carotene did not inhibit growth of either normal HKc or HKc/HPV16. In addition, HKc/HPV16 lines were more sensitive than normal HKc to modulation of keratin expression by RA and retinol. No differences were observed in the rate of uptake of [3H]RA or [3H]retinol between normal HKc and HKc/HPV16. Dot blot analysis of RNA extracted from HKc/HPV16 cultured in the absence or in the presence of 10(-7) M RA showed that the expression of the HPV16 open reading frames E6 and E7 is reduced 2- to 4-fold by RA. In addition, Northern blot analysis demonstrated that RA inhibition of E6 and E7 expression was both dose and time dependent. Overall, these results suggest that the increased sensitivity of the HKc/HPV16 lines to growth control by RA may be mediated by an inhibition of the expression of HPV16 gene products which are required for the maintenance of continuous growth.