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Cervical cancers are considered to originate from a series of pre-malignant lesions (cervical intra-epithelial neoplasia, CIN). The mechanisms behind these events are unknown. In addition to HPV infection, deletions of chromosome 3p have been found to be a frequent event in cervical cancer and likely play an important role in the transition of CIN to invasive cancer. To classify the potential role of 3p deletions in early-stage cervical carcinogenesis, we analyzed LOH of 3p in cervical precancers. Thirty cases with single or multiple CIN lesions were selected for the study, including 20 cases without and 10 cases with synchronous invasive cancers. Allelic losses on 1 or more 3p loci were recorded in 33% (3/9) of CIN II and 36% (5/14) of CIN III lesions from 20 cases without co-existing invasive cancer, whereas an increasing percentage of LOH was observed in the 10 precancerous lesions synchronous with invasive cancer, with 71% (5/7) CIN II and 76% (13/17) CIN III lesions. This result implies that 3p deletions have selective roles in early transition of pre-malignancy to invasive cancer. Comparing the LOH patterns between the 2 groups, genetic deletions in cases with invasive cancers involved extensive regions of 3p but were more localized in precancer cases without concomitant invasive cancer. Two interstitial regions, 3p22-21.3 around marker D3S1260 and 3p21.1 around markers D3S1289 and D3S1076, were most frequently deleted in both groups, suggesting that these 2 regions are novel tumor-suppressor loci which may play a role in early transition of cervical precancer to invasive cancer. Identical LOH patterns between multiple CIN lesions and synchronous invasive cancer in the same case suggests that different cervical precancers and invasive cancer are genetically linked and most likely originate from a single precursor cell., (Copyright 2000 Wiley-Liss, Inc.)

We microdissected 15 specimens of invasive cervical cancer co-existing with some of its precursors. Out of 15 cases, 10 carried HPV16, 2 HPV31, 1 HPV18 and 2 were HPV-negative. We found 3 HPV16 E6 variants among the 10 cases; one was A --> G in nt 131 (one case) and a second was A --> G in nt 276. The third, T;--> G in nt 350, was common, and was found in 5 of the 10 patients infected by HPV16. The type of HPV and the E6 variant were identical in all lesions within the same patient. Viral DNA present in normal epithelium was identical in type and E6 variant to HPV in the same patient's lesions. Multiple samples from invasive cancers with HPV were consistently positive. The data suggest that the originally infecting HPV, including its variant type in the E6 gene, persists unaltered in the whole series of CIN that precedes invasive cancer. Our data are compatible with an essential role of HPV manifested by persistence of the viral genome during the entire natural life history of cervical cancer. We did not confirm previous data on the specific association of invasive cancer with an HPV E6 variant (G at nt 350 rather than T). The discrepancy may depend on the relatively few cases investigated or selection of a special sub-set with progression from CIN to invasive cancer already manifest., (Copyright 1999 Wiley-Liss, Inc.)

Human papillomavirus (HPV) is probably a necessary but definitely not a sufficient cause of cervical carcinoma. However, it remains unclear which factors, in addition to HPV, are important for the development of cervical carcinoma and its precursor lesions. To address this issue, we conducted a case-control study nested in a population-based cohort consisting of women participating in cytological screening in one Swedish county, any time during 1969 through 1995. Detailed information on sexual practice, smoking habits and oral contraceptive (OC) use were collected through telephone interviews with 422 case patients diagnosed with cervical carcinoma in situ and 422 control subjects. All cytological smears were analyzed for presence of HPV 16/18 by a polymerase chain reaction (PCR)-based method. Odds ratios (OR) were used as measures of relative risk. After multivariate adjustment, a 2-fold higher risk was observed among current smokers compared with never smokers [OR 1.94; 95% confidence interval (CI 1.32-2.85)], an association apparently confined to women younger than 45 years. Current use of OCs was associated with a 4-fold increased risk overall (OR 3.64; 95% CI 1.91-6.93) with a monotonic increase with increasing duration of use (p for trend < 0.001). The number of sexual partners was significantly, positively associated with risk among HPV 16/18-negative (p for trend < 0.005) but not among HPV 16/18-positive women. Our data confirm the association between smoking and cervical carcinoma in situ, which might be age-dependent. Our results further indicate a relation with OC use and the risk for cervical carcinoma in situ.

Huge differences in incidence rates of invasive cervical cancer occur among populations. These differences reflect the influences of both etiological environmental factors and removal of precursor lesions detected upon screening. The purposes of this article are (i) to describe similarities and differences in the shapes and magnitudes of age-specific incidence rates of invasive cervical cancer before screening had an effect, (ii) to provide baseline data for further global study of screening effects, and (iii) to provide baseline incidence data for the design of optimal screening programs. To eliminate the impact of screening effects, we have selected age-specific incidence rates from times when and from populations in which screening was insignificant. The selected rates were suitably scaled and compared regarding age at onset of increase in incidence, age at peak incidence, and rate of subsequent decline. Despite a 16-fold difference in incidence rates, all curves had the same basic structure, with an increase to a peak followed by a decline or a plateau. Although all populations but one had an onset around age 25, 7 European countries showed an earlier peak age (mean = 46 vs. 59) and a more rapid decline after the peak than most other populations. The common basic shape of the age-specific incidence curve, overall, suggests a relatively similar development of invasive cervical cancer in different populations. These results illustrate the underlying similarities in the markedly different age-specific incidence rates of invasive cervical cancer. They also provide a basis for studying screening effects and for optimizing screening programs in specific geographic areas.

Specimens of squamous-cell neoplasms (81 invasive cancers, 36 in situ cancers, 70 dysplasias, 5 keratoacanthomas, 19 papillomas) and normal skin were immunostained with p53 antibody. Nuclear accumulation of p53 was visualized as following 2 distinct patterns: dispersed or compact. The former is interpreted as a reversible reaction to sunlight, whereas the latter, after microdissection and sequencing of DNA, has been shown to reflect clonal multiplication of keratinocytes with mutated p53. The dispersed pattern was diffusely distributed and usually only involved a small proportion of epidermal cells. The compact pattern was characterized as a contiguous area of homogeneously stained cells sharply demarcated from its surroundings. It involved patches of normal epidermis or large areas of dysplastic or malignant squamous epithelium. Immature cells were always stained, whereas immunoreactivity was variably present in differentiating keratinocytes. Dispersed patterns occurred in 94.7% of strongly UV-exposed skin (mainly face) and to a lesser extent in less exposed parts of the body. It showed no correlation to the age of the individual. About two-thirds of biopsies from individuals over age 50 displayed compact patterns in sun-exposed, otherwise normal, epidermis. About 65% of pre-malignant and malignant squamous-cell neoplasms had a compact pattern. The presence of p53 immunoreactivity as a compact pattern supports the idea that mutations of the p53 gene are early events in the sequence from dysplasia to invasive squamous-cell cancer of the skin. Also, even in the absence of cellular atypia, patches of epidermal cells can accumulate p53 in a way that is indistinguishable from that of cancer and pre-cancer.

It is known that down-regulation of cell surface platelet-derived growth factor (PDGF) receptors accompanies transformation by SV40. In this work human embryonic lung fibroblasts were used as a model system to study the effects of SV 40 on PDGF receptor expression. It is shown that transformation by SV 40 early region leads to a total loss of PDGF alpha-receptor and partial loss of beta-receptor mRNA. Microinjection experiments revealed that receptor down-regulation was a primary effect, and not only secondary to transformation and clonal selection. Total loss of PDGF alpha-receptor expression requires both large T and small t, and down-regulation of the PDGF alpha-receptor occurs independently of p53 and Rb binding to large T.

It has long been known that the growth rate of cells in vitro can be retarded by providing substrates of restricted area. Such experiments were performed with adhesive islets, made by depositing metals onto agarose layers through templates of various sizes. Since normal cells are unable to adhere to agarose, they become confined to the metallic surface. Using such haptotactic islets, we have studied the role of membrane ruffling and cell locomotion in the resistance of AG1523 human fibroblasts to growth factor-induced mitogenesis. Cells plated on small substrates, i.e., 2,150 microns 2 in area, initially showed vigorous ruffling, which was suppressed by 8 h after plating but had resumed again by 12 h. In contrast, cells on larger-size islets showed a rapid decline and stabilization of ruffling activity. When the growth rate was measured for single cells cultured on haptotactic islets, it was found to increase linearly from areas of 4,280 microns 2 up to 425,000 microns 2. Since the area needed to saturate the growth response was approximately 50-fold larger than the area occupied by a single cell, the growth inhibition was attributed in part to an interference with locomotion. The implication that locomotion provided positive input into growth control mechanisms was subjected to a direct test by evaluating the effect of nine polypeptide growth factors on the motility of serum-starved cells. All except TGF-beta 1 stimulated movement. Finally, the mitogenic effect of growth factors was measured by [3H]thymidine incorporation and found to be proportional to motile activities, as quantitatively assayed. We conclude that locomotion suppression is a factor in AG1523 cell resistance to growth factor-induced mitogenesis.

Some gliomas, melanomas and squamous carcinomas have large numbers of EGF receptors which could, in these cases, be used for targeting with toxic agents. We investigated whether EGF could be conjugated to dextran, which is a suitable carrier for toxic agents, without losing its ability to bind to the receptor. Dextran of 20 kDa molecular weight was activated with I-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) yielding highly active pyridinium-isourea derivatives. EGF was coupled to the activated dextran through the amino terminus and glycine was added to block residual activity. The EGF-dextran conjugate was, after purification on Sephadex G25 and Sephacryl 200 columns, tested for its receptor binding properties on human malignant glioma, U343MGaC12:6, cells. The conjugate inhibited binding of 125I-EGF in a competitive assay, showing that the binding was receptor-specific. Dextran conjugated with glycine, without EGF, had no inhibitory effect. The conjugate was radio-labelled either on the EGF part with 125I or on the dextran part with 3H-glycine, and the internalization patterns were compared to the internalization of 125I-EGF. The radioactivity of the conjugates remained cell-associated for more than 20 hr, regardless of whether the radioactivity was on the EGF or on the dextran part, while the radioactivity of unconjugated EGF rapidly disappeared from the cells. Most of the cell-associated radioactivity was, at all analysed time intervals, located intracellularly. Thus, it seems promising to use dextran, conjugated with EGF, as a carrier of, for example, toxic radioactive nuclides.

Meningioma is a generally benign tumor derived from arachnoid tissue. We have investigated the presence of functionally active PDGF-receptors on human meningioma cells in culture. Tumor samples were obtained from 3 surgically removed benign meningiomas and normal arachnoid tissue from an autopsy case. Binding studies were performed by using 125I-labelled recombinant PDGF-AA and PDGF-BB. Only 125I-PDGF-BB showed specific binding to all tumor-cell cultures after incubation of cells for 2 hr at 4 degrees C. Effects of PDGF-AA and PDGF-BB on DNA synthesis were measured as 3H-thymidine incorporation during 48 hr of labelling cells maintained in Eagle's minimum essential medium 0.5% fetal calf serum. PDGF-BB but not PDGF-AA stimulated DNA synthesis in all 3 tumor-cell cultures. Total cellular RNA was analyzed by Northern blotting and hybridization with a 32P-labelled human PDGF beta-receptor probe, and PDGF beta-receptor mRNA was found in both tumor and arachnoid cell cultures. Furthermore, PDGF beta-receptor mRNA was shown to be present in 2 meningioma biopsies and immunohistochemical staining revealed that PDGF beta-receptors are present in meningioma and arachnoid tissues in vivo. It appears that a possible way of maintaining human meningioma cell growth in vivo is through activation of PDGF beta-receptors.

Agglutination by two lectins, Concanavalin A (Con A) and Ricinus communis agglutinin (RCA), has been investigated in a human lymphoid cell system. The main conclusions of this study are: (1) no systematic correlation exists between the neoplastic state and sensitivity to Con A or RCA; (2) cells of neoplastic lines vary unsystematically in their surface properties as evaluated by Con A agglutination, with the possible exception that presence of Epstein-Barr virus (EBV) is associated with a high degree of agglutination and (3) cells of diploid lymphoblastoid lines and phytohemagglutinin (PHA)-stimulated lymphoctes agglutinate similarly and significantly better than unstimulated T- or B-lymphocytes. The relatively simple Con A agglutination assay can be used as an adjunct in classification of human lymphoid cell lines.

Over 200 established human hematopoietic cell lines of normal and malignant origin have been investigated by morphological and functional parameters. Employing morphology as the overriding parameter four types of lines were identified. (1) Lymphoblastoid cell lines, derived from normal and neoplastic hematopoietic tissue, were characterized by the wide morphologic flexibility of individual lymphoblastoid cells, constant association with Epstein-Barr virus (EBV), polyclonal derivation, differentiation for immunoglobulin production (secretion) and their diploids. (2) Lymphoma cell lines. This type of line was established at a high frequency from Burkitt's lymphoma and rarely from other types of lymphoma, but never from patients without malignancy or with non-lymphoma malignancies. Important characteristics were morphologic stereotypia within each line, monoclonal derivation, common but not obligatory association with EBV, variability in the expression of Ig synthesis (no production, or membrane bound Ig, or secretion) and aneuploidy. (3) Myeloma cell lines could only rarely be obtained from patients with myeloma. The basis for classification of these lines is their production of Ig identical to the myeloma protein in vitro. Other important distinguishing features were: plasma cell morphology, absence of EBV and aneuploidy. (4) The leukemia cell line (MOLT 4) was the only line with T-cell characteristics and was easily distinguished from the other types. Important characteristics were a typical surface ultrastructure, absence of EBV and absence of immunoglobulin production, Individual lymphoblastoid lines were in principle identical whereas each line of the other three types had its own characteristic profile. The phenotypic characteristics of the lymphoblastoid lines were very stable during prolonged serial cultivation. Only in a few cases were secondary chromosomal, functional or morphologic alterations noted. We conclude that EBV-carrying lymphoblastoid lines can be obtained from non-neoplastic precursor cells from healthy as well as from diseased individuals. Lymphoma, myeloma and leukemia lines are only obtained from the respective neoplastic tissue but generally at a low frequency. With the exception of Burkitt's lymphoma, malignant hematopoietic tissue and leukemia frequently give rise to established cell lines in vitro of the lymphoblastoid type rather than lines derived from the neoplastic cells;

The kinetics of aging of normal human diploid brain cells in culture have been determined using the miniclone technique in which cells are cloned in the presence of a large number of other cells. The miniclone technique records the behaviour of every viable cell in the sample, not merely those cells capable of forming visible clones. This technique permits the direct measurement of the reproductive potential of individual cells growing in bulk culture and of the dispersion of the sizes of colonies generated by dividing cells. The fraction of cells that are able to divide declines smoothly and continuously from the beginning of in vitro cultures of human glial cells. There is a broad distribution of colony sizes; even at the earliest passages there are significant numbers of small colonies. With increasing age of the culture there is a shift in the distribution, so that fewer large colonies and more small colonies occur. The distribution of intermitotic times is almost identical in young and middle-aged cultures. Our data seem to exclude quite positively any description in terms of a catastrophe or any abrupt change in the population. On the contrary, the decline in reproductive potential may be described adequately either as a linear change with time, or as predicted by the mortality theory of Shall and Stein (1979), in which the single constant, gamma, describes the change in reproductive potential over the entire lifetime.

Different types of human cells which normally grow as monolayers or suspension cultures were tested for their capacity to form and grow as spheroids. Sixteen out of the 27 tested tumour cell lines formed spheroids. Nearly all of these spheroids also grew. With only two exceptions the doubling times were longer when the tumour cells grew as spheroids than when they grew in conventional mass culture. Eleven out of 13 tested human non-tumour cells formed small spheroids but of these only the spheroids of lymphoid origin could grow. These lymphoid cells grew faster when aggregated to spheroids than when in single-cell suspension culture. None of the other non-tumour cells, which normally grew as monolayers, could grow as spheroids. The normally monolayer-cultured tumour cells formed symmetrical spheroids with smooth surfaces while the normally suspension-cultured cells formed irregular spheroids with rough surfaces. All large spheroids had a necrotic centre surrounded by a shell of viable cells. The thickness of the viable cell layer varied depending on cell type. The shape and organization of cells within the spheroids also varied largely. The results show that many types of human cells can be cultured as spheroids and that a wide spectrum of morphological appearances and growth rates can be obtained.