Desmosome development in an in vitro model.

A model has been devised to study the in vitro formation of desmonsomes. The model is based on the differential labeling of two subpopulations of a desmosome-forming human cancer line (C4I). The labeled subpopulations are dispersed, preincubated separately on a shaking water bath for 24 h to allow the internalization of desmosome fragments and the repair of the cell surface, and then mixed, and allowed to aggregate. Aliquots of the mixed suspension are fixed at various intervals. The time between mixing and fixation represents the maximum age of any junction between dissimilarly labeled cells. The beginnings of desmosome formation were observed within a few minutes after the beginning of aggregation. Close apposition of cell membranes was seen immediately after mixing, followed within 15 min by the appearance of a submembrane density in one or both of the interacting cells. Intracytoplasmic filament formation takes place at between 15 and 30 min. Desmosome formation is complete by 90 min. The process is accompanied by a progressive widening of the extracellular space and the desification and organization of the extracellular material and the submembrane plaques.

The mystery of chromosomal translocations in cancer.

Chromosomal translocations in human cancer may result in products that can be suppressed by targeting drugs. An example is bcr-abl tyrosine kinase in chronic myelogenous leukemia that can be treated with imatinib mesylate. However, the mechanisms of translocations or exchanges of chromosomal segments are virtually unknown. In this summary, chromosomal translocations in human cancer are compared with 'crossing over' of chromosomal segments occurring during the first meiotic division. Several proposed mechanisms of the exchange of DNA between and among chromosomes are discussed. The conditions that appear essential for these events to occur are listed. Among them are proximity of the involved DNA segments, mechanisms of excising the target DNA, its transport to the new location, and integration into the pre-existing chromosome. The conclusion based on extensive review of the literature is that practically nothing is known about the mechanism of 'crossing over' or translocation. Based on prior work on normal human cells, it is suggested that only one of the two autosomes participates in these events that may include loss of heterozygozity, another common abnormality in human cancer.

Distribution of T-lymphocytes and B-lymphocytes in peripheral blood and effusions of patients with cancer.

The relative distribution of T- and B-lymphocytes in the blood and in pleural or abdominal effusions was compared among 24 patients with fluid accumulation due to metastatic cancer and 8 patients without evidence of cancer. The data obtained indicated that the mean percentage of T-lymphocytes in malignant effusions was significantly greater than that in the peripheral blood of the same patients. At the same time, the mean eprcentage of B-lymphocytes was decreased in malignant effusions when compared with peripheral blood. Neither of these differences was observed when effusions and blood of patients with nonmalignant effusions were compared. In addition, patients with both types of effusions had fewer total lymphocytes in their blood than did normal control patients, whereas those with cancer-associated effusions had an increased proportion of active T-lymphocytes in their blood.

DNA distribution in human colon carcinomas and its relationship to clinical behavior.

Striking differences in outcome of surgically treated colon adenocarcinomas were observed in 33 patients according to the DNA distribution patterns in tumor nuclei, measured by flow cytometry. As discussed and defined in the text and confirmed by appropriate control studies, the tumors were classified into 2 groups: predominantly diploid (20 tumors) and predominantly nondiploid (13 tumors). During the follow-up period of 3-5 years, 12 of the 13 patients with "nondiploid" tumors died of disease within 4-34 months, and the 1 patient still alive after 59 months has extensive metastases. Only 6 of the 20 patients with "diploid" tumors died of disease, sometimes after a slow, protracted clinical course. There were 14 patients with no evidence of disease for periods ranging from 30 to 60 months; 1 of these patients died of other causes. Histologic grading and Dukes' staging appeared to play a relatively limited role in the outcome, although lymph node metastases and vascular invasion were more often observed in the nondiploid tumors. These observations suggest that the determination of DNA distribution in colon carcinomas may prove to be of prognostic value.

Nuclear pores and DNA ploidy in human bladder carcinomas.

The number of nuclear pores per sq micron was determined on the freeze-fractured nuclei of 20 human bladder tumors and five control samples of normal bladder epithelium. Measurements of the nuclear surface and volume were also performed, and the mean number of pores per nucleus and the ratio of pore to volume were calculated. The DNA distribution pattern on the same samples was determined by flow cytometry. All control samples and 12 tumors were diploid, and eight tumors were aneuploid. The mean number of pores per sq micron and mean total number of pores per nucleus in the control samples and in diploid tumors were similar. In the aneuploid tumors, both values were significantly higher. However, the ratio of pore to volume was shown to be constant regardless of the DNA content. It was further observed that, in aneuploid tumors, there are two populations of nuclei, one with density of pores similar to the diploid tumor and one with a higher pore density. Because aneuploid bladder tumors have been shown to have more aggressive behavior than diploid tumors, increased density of nuclear pores or their total number per nucleus may be related to tumor behavior. This view is supported by the observation that five of eight tumors with increased density and total pore number were invasive, while all tumors with low pore number were noninvasive.

Expression of Ca antigen in relation to cell cycle in cultured human tumor cells.

In order to explain the heterogeneity of Ca antigen (Ca) expression observed previously on human malignant cells, a relationship of antigen content with events in the cell cycle was studied by multiparameter flow cytometry on four continuous human cell lines, HeLaS3, C4l, HT29, and T24, grown exponentially in culture. In the four cell lines studied, there was an increase in Ca expression during the G1 and G2 + M phases whereas, during the S phase, the Ca expression was relatively constant. Although the level of Ca varied among the four cell lines, the mean amount of Ca expressed during the G2 + M phase was from 58 to 94% higher than the mean for the G1 phase. The G1 and G2 + M cell populations also displayed considerably greater variability of Ca content when compared with cells in the S phase. The level of Ca among cell lines appeared to have an inverse relationship to DNA content expressed as DNA index. Ca expression appeared to be related to RNA content and hence, presumably, ribosomal content, across the cell cycle. This study suggests that some of the heterogeneity of Ca expression in human cancer cells may be related to cell cycle events.

Modification of Ha-ras oncogene p21 expression and cell cycle progression in the human colonic cancer cell line HT-29.

Multiparameter flow cytometric measurements of the Ha-ras oncogene product, Ha-p21, versus DNA content were used to study the effect of prednisolone, sodium butyrate, and hyperosmolality on the expression of this gene during the cell cycle of HT-29, a human colonic carcinoma cell line. In control cells the expression of Ha-p21 was cell cycle dependent; it increased during G1 and remained approximately constant as cells traversed the S- and G2 + M phases. Two compartments of G1 cells, one expressing low (G1A) and the other (G1B) high levels of Ha-p21 could be identified. Cells grown with prednisolone (1.4-2.1 microM) expressed higher Ha-p21 levels than controls. Cell cycle analysis revealed that this effect was accompanied by a change in the distribution of cells in G1 phase: whereas the proportion of cells in G1A was reduced, that of cells in G1B was increased. The steroid had no detectable effect on cells in S and G2 + M. By contrast, sodium butyrate and hyperosmolality caused a marked decrease in Ha-p21 content. This reduction was not accompanied by any modification of the proportion of cells in the cell cycle compartments. These results would suggest that Ha-p21 is not likely to be a primary regulator of cell cycle progression in HT-29 cells.

Induction of alkaline phosphatase activity in cultured human intracranial tumor cells.

Alkaline phosphatase activity in several cultured primary human intracranial tumor cells varied over a relatively wide range, and there was no correlation between specific activity and the type of tumor from which the cultures were derived. The enzyme was thermolabile, and its activity was strongly inhibited by l-bromotetramisole, levamisole, and L-homoarginine but not by L-phenylalanine and L-phenylalanyglycylglycine. These are the characteristics of the liver-bone-kidney form of alkaline phosphatase. Prednisolone induced increased levels of enzyme activity in most cultures, and sodium butyrate acted as an inducer in cultures of pituitary adenoma and hemangioblastoma cells. The increase was most pronounced when response cells were exposed to both stimuli simultaneously. The induced alkaline phosphatase had the same properties as the enzyme of cells grown in the absence of inducers. Increased alkaline phosphatase activity was not induced by osmolality changes of the culture medium; this feature appears to be characteristic of cells producing the liver-bone-kidney enzyme form.

Chromosome analysis and alkaline phosphatase of C41, a cell line of human cervical origin distinct from HeLa.

The C41 cell line, which was derived from a human squamous carcinoma of the uterine cervix, has been characterized by analysis of quinacrine-banded metaphase chromosomes and study of alkaline phosphatase. C41 cells have a distinctive karyotype. They are hypodiploid, with a highly characteristic series of marker chromosomes, most of them derived by translocation or deletion. They contain no HeLa cell marker chromosomes, and the cell line shows no evidence of HeLa cell contamination. Nevertheless, the C41 and the HeLa cell line, both derived from cervix cancer, although of a different histological type, produce similar alkaline phosphatases. The enzyme is heat stable (placental type), is inhibited by L-phenylalanine, and responds to the inducing effects of prednisolone and/or hyperosmolality.

Protein expression in relation to the cell cycle of exponentially growing human prostatic epithelial cells.

This report concerns the study of the relationship between protein expression and the cell cycle in exponentially proliferating benign and malignant human prostate epithelial cells in short-term cultures. Multiparameter flow cytometric measurements were performed to correlate the expression of prostate-specific acid phosphatase, epithelial membrane antigen and epitectin with cell cycle progression. The expression of the three proteins was heterogeneous in G1 cells. The early post-mitotic cells exhibited the lowest levels when compared with late G1 cells, wherein the expression was many times greater. There was no further increase as the cells progressed through S and G2 + M. These findings, corroborating prior observations in other systems, suggest the possibility that the levels of the proteins studied increase during the G1 phase of the cell cycle and drop during or immediately after cytokinesis. As an alternate explanation, the heterogeneity of protein expression characteristic of G1 cells may be due, at least in part, to an asymmetric apportionment of cell constituents at mitosis.

Cervical and vaginal squamous cell abnormalities in women infected with human immunodeficiency virus.

We sought to determine whether women infected with human immunodeficiency virus (HIV) had cervicovaginal cellular changes suggesting lower genital tract neoplasia or human papillomavirus (HPV) infection at a rate different from that in women without HIV infection. In a blinded fashion, cytological preparations of cervicovaginal smears from women infected with the HIV were analyzed and compared to preparations from women at high risk for but not infected with HIV. Eleven of 35 (31%) HIV-infected subjects had evidence of squamous abnormalities compared with 1 of 23 (4%) non-HIV-infected women (p = 0.019). Nine of 35 (26%) HIV-infected women had cytohistological evidence of human papillomavirus (HPV) infection compared to 1 of 23 (4%) non-HIV-infected women (p = 0.072). We conclude that HIV-infected women have a high prevalence of cervical and vaginal cytological abnormalities and evidence of genital HPV infection. Further study is necessary to determine whether there is an increased risk for cervicovaginal neoplastic disorders in women infected with HIV.

Aspiration biopsy--a tool in surgical pathology.

A historical overview of the origins of the thin needle aspiration biopsy is given. Current applications of this technique to palpable lesions and lesions requiring various forms of imaging techniques are briefly summarized. The principal advantages of this method of diagnosis are its rapidity, versatility, and applicability to patients with palpable lesions as an office procedure. The procedure is nearly painless, hence readily accepted, and often shortens the anxious wait for a diagnosis. In competent hands, given an adequate sample, the diagnosis can be rendered with an accuracy approaching that of a frozen section. In many instances of intrathoracic and intraabdominal space-occupying lesions, exploratory thoracotomies and laparotomies can be avoided, thus reducing significantly the time of hospitalization. The procedure is safe. Seeding of cancer along the needle track has been reported in a few instances, but the risk of this event must be considered extremely low. Thin needle biopsy may offer the patient an opportunity to participate in therapeutic decisions, for example, in women with breast cancer. Finally, the aspirated sample offers a number of research options that may lead to a better assessment of prognostic parameters and response to treatment in cancer.

Condylomata acuminata of the urinary bladder. Natural history, viral typing, and DNA content.

Three patients with condylomata acuminata of the urinary bladder are reported. Two of the patients were immunosuppressed, and one had longstanding extensive condylomata acuminata of the external genitalia and adjacent areas. All lesions recurred at least once and were difficult to treat. The diagnosis was confirmed by in situ hybridization on archival material with human papillomavirus (HPV) DNA probes under stringent conditions. In two of the patients, probes for HPV types 6 and 11 were positive; HPV 11 only was identified in one patient. Probes for HPV types 16 and 18 and pBR322 vector controls were negative. In one patient with a strong hybridization signal, the lesion was also positive for common papillomavirus antigen. DNA content measured by cytophotometry of Feulgen-stained whole nuclei isolated from lesions in two patients revealed a markedly aneuploid DNA pattern. Whether this is a factor in the behavior of the lesions is not known at this time. Although rare, HPV infection of the urinary bladder may result in widespread condylomatosis and may mimic giant condylomas of Buschke-Löwenstein or even verrucous carcinomas, sometimes necessitating radical treatment. Nevertheless, until there is proof to the contrary, the lesions must be considered benign and should not be confused with squamous cancer of the bladder.

Pseudosarcomatous stromal reaction in primary and metastatic urothelial carcinoma. A source of diagnostic difficulty.

We report five urothelial carcinomas (one primary and four metastatic) with pseudosarcomatous stromal reaction. The exuberant stromal reaction led to a histologic misdiagnosis in three of the original small biopsy specimens. The differential diagnoses of primary spindle cell lesions of urinary tract include spindle cell carcinoma, carcinosarcoma, sarcoma, and benign pseudosarcomatous lesions. The distinction between those conditions and urothelial carcinomas with pseudosarcomatous stromal reaction is obviously of great clinical significance. In an initial small biopsy specimen, it may be difficult to make such a distinction. Immunostaining for cytokeratin and examination of more material should be performed. In our study of metastatic urothelial carcinomas with pseudosarcomatous stromal reaction, clinicopathologic correlations along with immunostaining for keratin proved to be useful. The stromal inductive capability of transplanted urothelium, which has been established in animal experiments, has occasionally also been reported in humans. The malignant urothelium in humans similarly appears to be capable of inducing stromal reaction.

Flow-microfluorometric analysis of nuclei isolated from various normal and malignant human epithelial tissues. A preliminary report.

In order to obviate some of the technical problems associated with preparation of monocellular cell suspensions required for flow fluorometry, isolation of nuclei from several types of benign and malignant human tissues was undertaken. Satisfactory preparations of nuclei were obtained from epithelia of the uterine cervix and colon and from lung tissue using the citric acid method. The sucrose method was effective with colonic epithelium only. Distribution of deoxyribonucleic acid content in these nuclei was measured based on green fluorescence of acridine orange and red fluorescence of propidium iodide in a Bio-Physics Cytofluorograph. Essentially diploid patterns of deoxyribonucleic acid distribution were observed for all benign samples regardless of tissue origin whereas the malignant samples gave histograms suggestive of abnormal deoxyribonucleic acid distribution. Preliminary observations on distribution of single-stranded nucleic acids using acridine orange red fluorescence showed marked differences between populations of benign and malignant nuclei. Isolated nuclei appear to be suitable for flow-through microfluorometric analysis and offer some significant advantages over intact cells.

In situ hybridization with human papillomavirus using biotinylated DNA probes on archival cervical smears.

We report a method of in situ hybridization (ISH) of 10-year-old archival cervical smears with a cocktail of nick-translated human papillomavirus (HPV) DNA types 6, 11, 16, 18, and 31. The method, which does not require destaining, results in excellent preservation of morphological detail with only 2% cell loss. Methods of smear treatment and detection of the biotinylated probe with a multistep avidin-biotin-immunoperoxidase method are described. Biotinylated PBR 322 plasmid and biotinylated human DNA were used as negative and positive controls in each run. Twenty-nine of 50 smears (58%) showing changes consistent with CIN I-II were positive for HPV. Fourteen corresponding cervical biopsies were also studied by ISH, seven corresponding to HPV-positive smears and seven to HPV-negative smears. HPV DNA was demonstrated in six of seven biopsies (87%) from the positive group but none could be demonstrated in the negative group. We conclude that retrospective study can be performed on routine alcohol-fixed, Papanicolaou-stained cervical smears with biotinylated HPV probes with excellent cell preservation, minimal cell loss, and high degrees of specificity.

Quantitation of oncogene products by computer-assisted image analysis and flow cytometry.

The use of antibodies permits the study of oncogene product expression in cells and tissues. However, quantitation of the levels of expression in immunohistochemical preparations is beset by difficulties, and the available scoring system provide semiquantitative data at best. Here we describe the use of computer-assisted image analysis for determination of oncoprotein levels in a model system and compare the results with those generated by flow cytometric analysis. The oncogene products measured are located in the nucleus (c-myc p62 and c-fos p55), the inner surface of the membrane (c-ras p21), and both sides of the membrane (c-erbB-2 p185). In each instance, both analytic modalities yielded concordant results. Our data indicate that computer-assisted image analysis is a useful tool for quantitating cell components in immunohistochemical preparations.

The use of a slide spinner in the analysis of cell dispersion.

A simple and reliable method of determining the degree of dispersion of a cell suspension has been developed using the Perkin-Elmer Uni-Smear Spinner. Optimum conditions regarding rate and duration of spin, etc., were first ascertained using dispersed cell cultures including human cervical cancer cells as well as gynecologic samples. After spinning, single cells in suspension appeared as isolated cells on the slides. Cell aggregates, on the other hand, remained together. Therefore, the distribution of cells in various sized aggregates could be easily quantitated and the slides retained for future review. This method was used to evaluate the dispersing effects of trypsin, ethylenediaminetetraacetate and and syringing human on human gynecology samples obtained by routine cervical scrapes. None of the dispersion methods has, so far, produced an adequate monodispersed cell suspension without unacceptable cell loss.

Mapping of the urinary bladder: its impact on the concepts of bladder cancer.

The results of mapping of the urinary bladder are reported and discussed. It has been shown that Brunn's nests, cystitis cystica, and the vaginal type of squamous metaplasia are commonly found in normal bladders and thus cannot be considered as precancerous lesions. Mapping of cancerous bladders and related histologic and clinical observations strongly suggests that there are two distinct pathways in bladder neoplasia: the papillary pathway and the nonpapillary pathway. Papillary tumors with thin stalks must be considered as a focal expression of the proliferative potential of the urothelium. They are per se quite harmless but may be followed by other manifestations of neoplasia. Broad based papillary tumors, regardless of grade, may have "pushy borders," which extend into the lamina propria but rarely invade the muscularis. Nonpapillary flat lesions, notably atypical hyperplasia and carcinoma in situ, appear to be the principal source of invasive and metastasizing bladder cancer. The presence of the flat lesions puts the patient at high risk for the development of invasive carcinoma. Methods of assessment of the cancerous bladder are suggested and discussed.

Flow cytometric measurements of DNA and other cell components in human tumors: a critical appraisal.

Fundamental principles of flow cytometry with emphasis on DNA measurements and cell cycle analysis in human cells and tissues are summarized. Some of the pitfalls of cell preparation techniques and histogram interpretation are discussed at length. While consensus has been reached for some organs and tumors that DNA quantitation by flow cytometry (or image cytometry) may be of prognostic value, for most cancers studied to date the information remains incomplete. Thoroughly lacking are well-structured prospective studies because retrospective studies, while suggestive, may not necessarily be of the same value. Potential usefulness of other tumor markers is briefly discussed. Many fundamental questions concerning definitions of "diploid" and "aneuploid" tumors have not been satisfactorily settled. While the goal of "objective measurements" is worthy of further pursuit, the interpretation of results is often highly subjective. The biologic reasons for behavioral differences between diploid and aneuploid tumors are still totally obscure.