Polymorphism at codon 72 of p53 is not associated with cervical cancer risk.

P53 allelic polymorphism at codon 72 has been studied as a possible predisposing factor for cervical carcinogenesis with inconsistent results. Storey and colleagues recently published the interesting finding of a 7-fold increased risk for cervical cancer in women homozygous for the arginine allele at codon 72. This stimulated a number of independent investigations, the majority of which found no association of cervical cancer and arginine homozygosity. With the use of a modified Storey method for determining codon 72 allelotypes, DNA was examined from 431 microdissected, formalin-fixed, archival cervical conization specimens ranging from low-grade squamous lesions to invasive cancer. An alternative independent method using restriction fragment length polymorphism analysis was performed on all arginine homozygotes and all indeterminate cases for confirmation and final allelotype assignment. With the use of Storey's method alone, logistic regression suggested an association (odds ratio, 1.42) between arginine homozygosity and invasive disease. However, with the use of the combined method for accurate allelotyping, this trend disappeared (odds ratio, 1.00), the discordance was clearly resolvable as being due to methodologic variables. With the use of two separate methods for codon 72 allelotyping and accounting for a number of the issues raised in previously published reports, there is no increased risk for invasive cervical cancer associated with arginine homozygosity at codon 72 of p53.

Performance of liquid-based, thin-layer cervical cytology: correlation with reference diagnoses and human papillomavirus testing.

The performance of thin-layer cervical cytology with the use of ThinPrep (Cytyc Corporation, Boxborough, MA) was assessed by comparing the original independent diagnosis of ThinPrep slides and conventional smears prepared from 1780 split samples with the most abnormal diagnosis per patient on the basis of an independent pathologist's masked review and with the detection of cancer-associated types of human papillomavirus (HPV) DNA. Cases were selected on the basis of the original diagnoses to include all discordant pairs (those diagnosed as atypical squamous cells of undetermined significance or higher grade, n = 1017), all concordant abnormal pairs (n = 444), and a random 5% of concordant normal pairs (n = 319). In screening centers, thin-layer cytology detected 135 (70.3%) of 192 women diagnosed as having squamous epithelial lesions or a higher grade in the independent review, whereas locally read smears detected 91 (47.4%) of these patients (P < .001). In hospital-based cytology laboratories, thin-layer cytology detected 308 (86.3%) of 357 women diagnosed with SILs or a higher grade in the independent review, compared with 283 (79.3%) diagnosed with smears (P = .011). Cancer-associated types of HPV DNA were detected in a slightly higher proportion of women with smears diagnosed as SILs than in women with thin-layer cytology diagnosed as SILs, whereas the overall number of HPV-associated SILs diagnosed was higher with thin-layer cytology. These data suggest that the ThinPrep method detects a higher percentage of SILs as defined in a masked, independent review than do concurrently prepared smears and that diagnoses of SILs rendered with ThinPrep correlate with the detection of cancer-associated types of HPV.

Human papillomavirus and Epstein-Barr virus in sinonasal Schneiderian papillomas. An in situ hybridization and polymerase chain reaction study.

Using the polymerase chain reaction (PCR), it has been recently reported that the Epstein-Barr virus (EBV) is present in the majority of Schneiderian sinonasal papillomas (SNP) of the inverted type and may play a role in the pathogenesis of these lesions. The reported prevalence rates of human papillomavirus (HPV) in different types of SNP is also controversial and in need of clarification. Twenty-eight SNP from 27 patients were histologically classified and evaluated for evidence of EBV using PCR and 2 different sensitive and specific in situ hybridization (ISH) procedures for EBER1. Similarly, two methods of ISH were also used for the detection of HPV, using biotinylated DNA probes sensitive for 14 different HPV types as well as more sensitive and specific radioactive RNA probes for HPV types 6, 11, and 16. Polymerase chain reaction was successful in 19 papillomas, including 12 of 19 inverted SNP, 1 of 1 inverted SNP with squamous cell carcinoma, 4 of 5 fungiform SNP, and 2 of 3 oncocytic lesions. Southern blot hybridization of PCR products showed the presence of EBV DNA in two lesions, including one inverted SNP and the single inverted SNP with squamous cell carcinoma. By both DNA- and RNA-mRNA ISH, positivity for EBER was detected in rare stomal lymphocytes but not the overlying epithelium in the inverted SNP with SCC. The remaining cases, including the other inverted SNP positive for EBV by PCR, were completely negative by both ISH techniques. Human papillomavirus was detected by ISH in 1 of 19 (5%) inverted, 1 of 1 (100%) inverted with squamous cancer, 5 of 5 (100%) fungiform, and 0 of 3 (0%) oncocytic SNP. Three SNP contained HPV 6 (all fungiform), three SNP labeled for HPV 11 (two fungiform and the inverted SNP with squamous cancer), and one inverted SNP contained HPV 16. Of the five fungiform SNP, four showed foci of koilocytosis. The results indicate that EBV is not present in sinonasal papillomas. The presence of EBV positive stromal lymphocytes in these lesions may account for a proportion of PCR-positive cases. Oncocytic SNP are unassociated with HPV, whereas inverted SNP contain HPV in a minority of cases. In contrast, fungiform SNP are consistently associated with HPV types 6 and 11 and usually show histologic evidence of viral infection.

Influenza hemagglutinin assumes a tilted conformation during membrane fusion as determined by attenuated total reflection FTIR spectroscopy.

Fusion of influenza virus with target membranes is mediated by an acid-induced conformational change of the viral fusion protein hemagglutinin (HA) involving an extensive reorganization of the alpha-helices. A 'spring-loaded' displacement over at least 100 A provides a mechanism for the insertion of the fusion peptide into the target membrane, but does not explain how the two membranes are brought into fusion contact. Here we examine, by attenuated total reflection Fourier transform infrared spectroscopy, the secondary structure and orientation of HA reconstituted in planar membranes. At neutral pH, the orientation of the HA trimers in planar membranes is approximately perpendicular to the membrane. However, at the pH of fusion, the HA trimers are tilted 55-70 degrees from the membrane normal in the presence or absence of bound target membranes. In the absence of target membranes, the overall secondary structure of HA at the fusion pH is similar to that at neutral pH, but approximately 50-60 additional residues become alpha-helical upon the conformational change in the presence of bound target membranes. These results are discussed in terms of a structural model for the fusion intermediate of influenza HA.

Reconstitution of membrane fusion sites. A total internal reflection fluorescence microscopy study of influenza hemagglutinin-mediated membrane fusion.

Influenza hemagglutinin (HA, strain A/PR/8/34) was purified and reconstituted into supported planar membranes in a two-step process: 1) HA was purified by C12E8 detergent solubilization followed by detergent removal with Biobeads; (2) the purified HA was then incorporated into "viroplanes," i.e. supported planar membranes which contained the viral membrane proteins. This step was accomplished by a spontaneous reaction of the HA-proteoliposomes with a phospholipid monolayer that was supported on a quartz microscope slide. The reconstitution of the HA into the planar membranes was followed by total internal reflection fluorescence microscopy (TIRFM) using fluorescein-labeled HA. By changing the solution concentration of HA, surface concentrations between 2.4 x 10(4) and 4.3 x 10(4) HA monomers/micron 2 were reached. Greater than 90% of all HA molecules were oriented with their ectodomain facing away from the substrate toward the large aqueous compartment of the measuring cell. Binding experiments with conformation-sensitive monoclonal antibodies against HA established that the reconstituted HA could undergo the low pH-induced conformational change in the supported bilayer. Binding of vesicles containing the fluorescent lipid analog N-(7-nitro-2,1,3-benzoxadiazol-4-yl)egg phosphatidylethanolamine was also measured by TIRFM. Vesicle binding was promoted when sialic acid-containing gangliosides or negatively charged lipids were included in these target membranes. Membrane fusion of the HA bound vesicles was monitored by measuring long range (over several micrometers) lateral diffusion coefficients of the lipids in the bound layer by fluorescence recovery after photobleaching. The vesicles did not fuse at pH 7.4, but efficient vesicle fusion occurred on the viroplanes after acidification of the environment with pH 5 buffer. This fusion reaction was only observed when the bound vesicles exceeded a critical threshold surface concentration. The successful reconstitution of membrane fusion sites in a planar supported membrane system opens new possibilities for studying fusion intermediates by localized spectroscopy and microscopy.

Further studies on growth factor production by the TC-1 stromal cell line: pre-B stimulating activity.

Adherent murine stromal cells support long-term in vitro lymphopoiesis or myelopoiesis dependent on the culture conditions used. A cell line, TC-1, isolated from long-term liquid murine marrow cultures under conditions approaching those permissive for lymphoid growth, has been found to produce an activity that acts synergistically with interleukin-3 (IL-3) or colony-stimulating factor-1 (CSF-1) to stimulate in vitro myeloid colonies, but which has no intrinsic colony-stimulating activity. We report here the presence of multiple growth factors in conditioned medium (CM) from the TC-1 line, including granulocyte-macrophage colony-stimulating factor (GM-CSF) (bioassay with antibody blocking and messenger RNA [mRNA] analysis), granulocyte CSF (G-CSF) and IL-4 (factor-dependent cell line bioassay), and CSF-1 (radioimmunoassay, mRNA) along with a pre-B cell inducing activity, which appears separate from these CSFs and segregates with the myeloid synergizing activity through anion exchange, sizing, and Conconavalin A chromatography. Because these activities are not yet purified to homogeneity, their identity or lack of identity remains an open question. Assays of TC-1 CM or cellular mRNA analysis have given negative results for IL-1, IL-2, IL-3, IL-6, and IL-7, and IL-6 does not stimulate pre-B cells in this assay. However, IL-4 and G-CSF do stimulate in vitro induction of pre-B cells from pre-B and B-cell-depleted Balb/C marrow and are present in CM by selective cell line assay. A monoclonal antibody to IL-4 that inhibited its pre-B inducing activity did not inhibit pre-B inducing activity of TC-1 CM. These data suggest the existence of a unique synergizing and pre-B inducing factor(s) in TC-1 CM. Given the known capacity of subliminal levels of growth factors to act synergistically, an alternate possibility is that these biologic phenomena represent the actions of low concentrations of growth factors acting synergistically and possibly associated with some core protein.

Multilineage synergistic activity produced by a murine adherent marrow cell line.

We reported previously that a cell line (TC-1) derived from adherent marrow cells produced colony-stimulating factor 1 (CSF-1) and a separate activity that acts synergistically with CSF-1 to stimulate giant macrophage colonies. We now report that an activity in TC-1 conditioned media (CM) separate from CSF-1 also synergizes multilineage colony formation by pure interleukin 3 (IL 3) and a crude source of granulocyte-macrophage colony-stimulating activity (GM-CSA) (murine lung-conditioned media). IL 3-induced megakaryocyte colony formation is also synergized. The CSF-1-dependent synergistic activity is not blocked by antibodies to IL 3 and is characterized as a nondialyzable (mol wt cutoff 3,000), heat-stable (56 degrees C, 30') activity that binds to DE-52 cellulose under conditions in which IL 3 does not. This material has an apparent mol wt of approximately 200,000 by Sephadex G100 chromatography, and the bulk of it binds to Concanavalin A (Con A) and elutes off with alpha-methyl mannoside, indicating that it is a glycoprotein. As reported separately, these purified active fractions also have a pre-B cell-inducing activity. In addition, a non-IL 3 activity stimulates proliferation of the factor-dependent cell lines FDC-P1 and DA-1. These data indicate that an adherent marrow cell line produces a growth factor(s) that synergizes with IL 3, GM-CSA, and CSF-1 and induces pre-B cell formation. This may be an important regulator of early multilineage lymphohemopoiesis.

Stromal cell regulation of lymphoid and myeloid differentiation.

In vitro microenvironmental influences seem to be critical for both B lymphocyte and myeloid differentiation. Studies on murine Dexter cultures and Whitlock-Witte lymphocyte cultures suggest the presence of two critical stromal regulatory cells: an alkaline-phosphatase-positive epithelioid cell and a macrophage. Further data suggest that these cells are capable of producing colony stimulating factor-1, granulocyte-macrophage CSF, a myeloid synergizing activity, and probably separate B cell growth factors. Isolation of a cell line from Dexter stroma was accomplished and this line produced CSF-1, GM-CSF, a pre-B cell and myeloid synergizing activity, and an activity acting on differentiated B cells. We speculate that the Dexter and Whitlock-Witte in vitro culture systems are regulated by factors produced by the two adherent cell types. A lineage nonspecific factor capable of inducing cells into the B lineage or synergizing with interleukin-3, GM-CSF, and CSF-1 is produced, which presumably acts on early stem cells. In addition, the cell line produces GM-CSF, CSF-1, and a factor acting on differentiated B cells. We speculate that in these culture systems, these "terminal differentiating hormones" regulate the final pathway of differentiation, whereas the pre-B-synergizing activity supports early stem cells that can then respond to the other differentiating hormones.

Studies on the regulation of hemopoiesis.

Normal hemopoietic cell differentiation and proliferation is critically dependent upon marrow stromal elements. Long-term liquid culture of marrow provides a model for the study of stromal function. We evaluated the effects of radiation and 5-fluorouracil (5-FU) on various aspects of long-term murine hemopoietic cell growth and stromal function. Exposure of C57BL/6J murine adherent cells from long-term marrow cultures to varying doses of irradiation (0-1000 rad) in vitro resulted in the elaboration of growth factors stimulating granulocyte, macrophage, megakaryocyte, mixed megakaryocyte-granulocyte macrophage, and blast colonies. This increased production of growth factors appears to be related to the ablation of normal granulocyte production in the culture system since addition of normal stroma to irradiated stroma blocks growth factor production. Two cell types appear to be mediating stromal factor production and support of liquid culture hemopoiesis: a macrophagelike cell and an alkaline-phosphatase-positive epithelioid cell. Exposure of these two cell types to pokeweed mitogen results in marked enhancement of growth factor production. Furthermore, a cell line isolated from normal murine stroma produced an activity capable of acting at an early hemopoietic stem cell level and of inducing secondary marrow cell lines. The establishment of cultures from 5-FU-treated animals revealed that chemotherapy-depleted marrow was capable of establishing adequate stromal function and that the residual surviving stem cells had a higher than normal proliferative rate. In addition, the function of granulocytes derived from this post-5-FU marrow was normal. Thus, it appears that both chemotherapy and radiation exposure of marrow results in an enhanced capacity of stromal elements to produce growth factors and support hemopoiesis and that post-5-FU marrow represents an enriched source of high proliferative potential bone marrow stem cells.