Action of cytochalasin D on cells of established lines. I. Early events.

HeLa, Vero, L, HEp2, and MDBK cells respond immediately to 0.2-0.5 microg/ml cytochalasin D (CD) with sustained contraction (contracture), loss of microvilli, expression of endoplasmic contents (zeiosis), nuclear protrusion, and extension of cytoplasmic processes. The development of these changes is depicted, and the dose-response patterns in these cell lines are described. MDBK is generally most resistant and HeLa most sensitive to these effects of CD. Cells in G(1) are most sensitive to CD; responsiveness decreases progressively during early S and is least in mid S through G(2). CD inhibits transport of [(14)C]deoxyglucose in HeLa by about 45% but has no significant effect on hexose uptake in Vero and MDBK; sugar transport is thus apparently unrelated to any morphologic effect of CD. Although spreading and attachment are impeded, CD does not decrease and may even enhance the adhesiveness of established monolayers. Contraction appears to be a primary early effect of CD, upon which other visible changes follow. It is prevented by some inhibitors of energy metabolism (deoxyglucose and dinitrophenol) and does not occur in glycerinated models without ATP. The possible bases of the contractile response to CD are discussed. Although direct or indirect action of CD on some microfilaments may occur, a generalized structural disruption of contractile filaments by CD is considered unlikely.

Action of cytochalasin D on cells of established lines. III. Zeiosis and movements at the cell surface.

The projection of knobby protuberances at the cell surface (zeiosis) is a general cellular response to cytochalasin D (CD), resulting from herniation of endoplasm through undefended places of the cortex during cell contractions and displacement of microfilaments induced by CD. Zeiosis is prevented by agents that interfere with the contractile response to CD, such as inhibitors of energy metabolism or cyclic AMP. The developed protrusions, which remain relatively stable in the presence of CD, contain chiefly mono- or subribosomes, and occasionally other organelles normally resident in endoplasm; compact microfilament felt occupies their bases and extends into their proximal stalks. Protein synthesis in the knobs is less than half of that in the polyribosome-containing endoplasm residual in the main body of the cell. Knobs first protrude singly near the margin of the contracting cells and rapidly cluster into small groups in the periphery even at lower temperature. The clusters then migrate centripetally and coalesce into a large aggregate near the apex of the immobilized and retracted cell: this movement is energy- and temperature-dependent. Aggregation is more prominent and stable in cell lines of epithelial derivation than in fibroblastic or other lines in which nuclear extrusion occurs more readily. The latter is regarded as a special manifestation of zeiosis. Macromarkers, such as latex spherules, migrate like the zeiotic knobs on the cell surfaces in the presence of CD. The aggregated knobs, although persistent for days in the presence of CD, are rapidly recessed after withdrawal of the agent as ruffling is resumed and the cells spread. These movements are discussed in terms of current concepts of mobility of the cell membrane.

Limitations of DNA histogram analysis by flow cytometry as a method of predicting chemosensitivity in a rat renal cancer model.

Analysis of DNA histograms obtained from rat renal cancer cells stained with propidium iodide and submitted to flow cytometry revealed a tumor cell population with prominent 2C and 4C peaks and with usually less than 10% each of S-phase or hyper-4C cells. The presence of an increased proportion of 4C cells was found to depend on the age and size of the tumor nodule. Sampling replicate portions of the same tumor or different tumors of the same size and age frequently revealed highly variable 4C:2C ratios. Treatment of animals bearing this tumor with a single i.p. injection of cyclophosphamide (CY), under conditions known to reduce the tumor burden by 80% within 1 week, or with 5-fluorouracil (FUra), which is ineffective against this tumor, in many cases did not yield changes in DNA histograms that permitted one to distinguish the effective drug. Either no marked difference in histogram shape occurred after therapy, or FUra induced more striking differences in cell cycle position than did CY. In tumor generations with greater than 15% S-phase cells, treatment with CY resulted in multiple effects on DNA histograms. These included detecting increased numbers of moribund cells (hypo-2C), a decrease in 4C cells, and an increase in hyper-4C cells. These changes did not occur with the ineffective agent FUra. The tumors grown in vitro show no evidence of replication of 4C cells. The DNA histograms of late-log-phase cultures have a major 2C peak and a minor S plus G2 hump. Since neither the untreated tumor in vivo nor that grown in vitro has a major hyper-4C cell population, it is probable that the tumor stem cells are chiefly 2C (diploid-hyperdiploid). Treatment in vitro of late-log-phase cultures with CY or FUra produces DNA histograms which permit identification of the effective agent. After CY, a major part of the cell population was hypo-2C (moribund) cells.

A stable propidium iodide staining procedure for flow cytometry.

A propidium iodide (PI) staining procedure is described in which 50 micrograms/ml PI in 10(-2) M Tris, pH 7.0, with 5 mM MgCl2 is used to stain murine erythroleukemia cells (MELC) grown in suspension culture as well as single cell suspensions derived from rat kidney adenocarcinoma and human prostatic carcinoma. Specificity of staining of nuclear DNA is achieved by enzymatic removal of RNA using RNAse in the staining solution. Virtually identical histograms, with the same G1 peak height and closely similar coefficients of variation (CVs), are obtained using a wide range of RNAse concentrations on replicate samples of MELC if the incubation times are sufficiently prolonged when employing the lower enzyme concentrations. For 1 mg/ml RNAse on logarithmically growing MELC, 30 min incubation at 37 degrees C is needed to obtain a maximum G1 peak height and optimal CV and there is no significant change in the histogram if the incubation is prolonged to 4 hr. For every 4-fold decrease in RNAse concentration, the incubation time at 37 degrees C must be doubled to obtain the same maximal G1 peak height and optimal CV. Unfixed cell preparations, whether derived from suspension or monolayer cultures or from solid tumors, are stable for 2 or more weeks if stored at 4 degrees C between flow cytometric analyses and histograms are usually only minimally altered if the stained cell samples are stored for 1-2 months at 4 degrees C. Sample decay is associated with bacterial contamination. If sterile preparative techniques are used initially, subsequent contamination of the stained preparations may be minimized by adding sodium azide to the stained samples at 0.1% without influencing fluorescence intensity. Glycerine may be added to 10% and the samples slowly frozen for storage without altering DNA histogram shapes. The simplicity of sample preparation and the stability of the resulting stained cell samples makes this procedure suitable for repetitive comparative sampling of tissue and cell populations over prolonged time spans.

Flow cytometry as a predictive modality in prostate cancer.

Clinical staging and histologic grading do not have sufficient predictive value to determine the response to therapy of any given prostate cancer. A review of the findings from the largest prospective study of patients with localized and locally advanced prostate cancer and from retrospective flow cytometric studies of specific disease stages suggests that DNA flow cytometry offers additional prognostic information for this disease. However, for the individual patient, this added information may have limited value, since approximately 15% of those with diploid disease will experience disease progression within 5 years as compared with half of those with nondiploid disease. We have found that ploidy does not predict length of survival once prostate cancer becomes disseminated, nor does it predict those who will benefit from receiving definitive radiation therapy for localized prostate cancer. On the other hand, for those who have persistent tumor, we have frequently found increased ploidy abnormalities in the tumor sampled after radiation therapy and are currently correlating this finding with clinical outcome. We have also found that DNA flow cytometry can be used to predict tumor volume. For larger, grade-matched diploid tumors, there are significant increases in the proliferation of both the tumor and the adjacent benign tissue, which we take to be evidence of "field effects" in this disease. An even more obvious manifestation of the same phenomenon is seen in the occurrence of aneuploidy in benign tissue near high-grade, large-volume prostate cancer. It is concluded that DNA flow cytometry has much to tell us about the natural history and biologic behavior of prostate cancer.

Correlation of genetic and immunodetection of TP53 mutations in malignant and benign prostate tissues.

The prognostic value of the p53 gene (TP53), the most commonly mutated gene in human cancers, has been well established for several cancer types. However, because varying frequencies of TP53 mutations have been identified in prostatic adenocarcinoma (CaP) by genetic and immunohistochemical (IHC) studies, the role of TP53 in CaP tumorigenesis is currently unresolved. These experimental discrepancies could be caused by tissue heterogeneity within prostatic neoplasms, variations in experimental protocols, or other factors. Thus, the goal of this study was to develop a reliable IHC approach for the detection of p53 in archival prostate tissue. The authors evaluated four p53 antibodies, CM-1, 1801, DO-1, and DO-7, for their ability to reveal p53. They chose two reference CaP cell lines, 26 patient specimens (including eight benign prostatic hyperplasias (BPHs), 16 CaPs, and two lymph node metastases), one prostate and nine kidney cell lines for p53 analysis. The TP53 status of these samples was characterized using single-strand conformational polymorphism (SSCP) analysis of RNA/PCR products and sequencing. IHC detection of p53 was markedly enhanced by using the combination of microwave heat-induced antigen unmasking and a cocktail of the DO-1 and DO-7 antibodies. This approach identified 14 of 15 (93%) cell lines and patient samples having TP53 missense mutations in the exons 5 to 8 region. Of the 21 patient samples and cell lines that were either normal by SSCP or expressed p53 mutations that are not expected to stain, 18 (86%) were immunonegative. Because of this good correlation between molecular and IHC analysis, this approach may help to resolve the uncertainty about TP53 in CaP tumorigenesis.

Flow cytometry: role in monitoring transitional cell carcinoma of bladder.

The ability to detect and monitor the course of transitional cell carcinoma (TCC) of the bladder using DNA histograms obtained from flow cytometry was studied. Voided urine and barbotage specimens were collected from patients with active TCC or a past history of TCC. These specimens were submitted to routine cytologic and flow cytometric analyses. Samples were considered to be positive if they met one or more of three criteria: if they had aneuploid or tetraploid peaks, if the DNA index of the major G1 peak was shifted more than 10 per cent from that of diploid cells, or if 15 per cent or more of the cells fell to the right of the major diploid G1 cell population thereby constituting a significant hyperdiploid cell population. Using these methods for patients with active disease, the detection rate was 91 per cent. In patients with a past history of TCC, positive histograms preceded the appearance of visible tumor in one third of the cases. Flow cytometry proved to be an excellent way of following patients with a past history of TCC or of screening patients suspected of having active disease. Following this protocol, few biologically active tumors go undetected. However, in 112 patients without a history of bladder cancer, the false positive or suspicious rate was 38 per cent. Before flow cytometry can be recommended as a widespread screening method for patients thought to be at risk of TCC of the bladder developing, this suspicious group will have to be eliminated.

Fixation method useful for cytologic examination and DNA flow cytometry of exfoliated bladder cells.

Single-institution studies have shown that DNA flow cytometry is superior to routine cytologic evaluation of following patients for bladder cancer recurrence. For 15 urine and 15 bladder washing specimens, we evaluated a fixative employing methanol plus acetic acid (MA), freshly mixed 20:1 (vol/vol). Routine cytologic evaluation following Papanicolaou staining, and DNA flow cytometry were performed. Paired aliquots from the same washings and urines were processed as fresh spray-fixed samples and MA-fixed samples. The majority of the MA-fixed specimens showed good nuclear preservation when assessed for chromatin texture, presence of distinct nuclear envelope, and clarity of nucleolus, while only a minority of the fresh urine and washing samples showed these features. Cytoplasmic degeneration was seen only in fresh specimens. The presence of aneuploidy and the percentage of hyperdiploid cells could be reliably determined in the MA-fixed samples. This fixation protocol is recommended for the transport of urine and bladder washing specimens to centralized laboratories for both cytologic and flow cytometric evaluation.

Comparison of flow cytometry to routine testicular biopsy in male infertility.

We compared DNA flow cytometry to morphologic evaluation of routine testicular biopsies as methods of monitoring spermatogenesis. The study group consisted of 14 azoospermic men and 5 others who underwent testicular surgery unassociated with fertility problems. The findings for both studies were divided into three groups: normal, moderately abnormal, and markedly abnormal. Correlations between the findings from routine biopsy and flow cytometry were good. Of 9 patients having normal testicular morphology, 7 had normal ploidy classes by DNA flow cytometry while 2 had moderately abnormal histograms. Of 5 cases with moderately abnormal morphology, 1 had normal, 1 had moderately abnormal, and 3 had markedly abnormal ploidy distributions. In 5 cases described as Sertoli cell only, all DNA histograms were markedly abnormal, consisting almost exclusively of diploid cells. DNA flow cytometry of testicular biopsies and aspirates has been demonstrated to be a rapid, reproducible, and objective approach in evaluating the infertile male and is a promising method to investigate spermatogenesis in an outpatient clinic in lieu of formal testis biopsy.

Comparison of histologic and flow cytometric evaluation of cyclophosphamide-induced testicular damage.

This study evaluates the ability of DNA histograms obtained by flow cytometry to detect and quantify reversible alterations in spermatogenesis induced by cyclophosphamide, a known inhibitor of spermatogenesis. Evaluation of per cent of cells in each of the haploid (lc), diploid (2c), and tetraploid peaks (4c) as determined by flow cytometry in treated and control Balb/C mice over a six-week period, and comparison with routine histologic evaluation have led us to conclude that DNA histogram evaluation is a rapid and accurate means of identifying testicular damage and recovery. This technique may be useful in sequential monitoring of the effects of malignancy and/or treatments applied on spermatogenesis in young men.

p53 and bcl-2 immunohistochemical alterations in prostate cancer treated with radiation therapy.

OBJECTIVES: Radiation therapy is definitive treatment for localized prostate cancer. It causes cellular deoxyribonucleic acid (DNA) damage, which, if irreparable, results in apoptosis or programmed cell death. Overexpression of mutant p53 and/or bcl-2 proteins prolongs cell survival despite exposure to damaging agents. We examined whether abnormal expression of either gene could help to explain radiation therapy failures in prostate cancer. METHODS: Archival tissue from patients who had failed radiation therapy as treatment for prostate cancer was obtained before and after treatment. These cancer samples were examined immunohistochemically for accumulation of p53 and bcl-2 proteins. Comparison was made with specimens from patients who had no evidence of recurrent or persistent disease at least 3 years following radiation therapy. RESULTS: High rates of p53 immunopositivity were found in the prostate tissue from all groups studied. More patients who had failed radiation therapy were found to have bcl-2 immunopositive specimens than were those without evidence for recurrent disease (41% preradiation and 61% postradiation versus 8%, P <0.05). More patients who failed radiation therapy had both p53 and bcl-2 immunopositive prostate tissue than did those who were treated successfully (32% preradiation and 48% postradiation versus 8%). CONCLUSIONS: bcl-2 immunopositivity, with or without concomitant detection of p53, was found in significantly more cancers of patients who failed radiation therapy. Positive staining for bcl-2 may serve as a marker for determining the radiation sensitivity of a tumor and thus may help to guide treatment options. It is also notable that a high proportion of the prostate cancers examined were immunopositive for p53.

Evaluation of vasovasostomy candidates by deoxyribonucleic acid flow cytometry of testicular aspirates.

Flow cytometry can be performed on testicular aspirates of vasovasostomy candidates preoperatively. On the basis of ploidy ratios and debris components, DNA histograms can be classified as normal or abnormal. Using this method, the likelihood of the presence of sperm may be predicted.

PIP: Of the approximately 10,000 vasectomy reversals performed in the US, 40%-50% of cases do not result in pregnancy. At the time of vasovasostomy, testicular aspirates were taken from 42 men, with a mean age of 38.1 and a mean duration since vasectomy of 10.9 years. DNA histograms were collected from the aspirates by automated flow cytometry. The histograms were classified as normal or abnormal based on the percent of haploid, diploid, and tetraploid cells and on the amount of low-signal debris. 21 (54%) of the 39 men available for analysis had normal ploidies, sperm present at the time of operation, and approximately 5.5% debris. Of the 14 men who had no sperm in the vasal fluid, 11 had abnormal histograms, showing abnormal distribution of ploidies; 5 (35.7%) had high levels of cellular debris. Normal distribution of ploidies, as shown by DNA flow cytometry, and low levels of cellular debris correlate with presence of sperm. Testicular parenchymal aspiration, therefore, provides a rapid, reproducible method for evaluating male fertility status after vasectomy reversal.

Testicular torsion: temporal considerations.

Unilateral testicular torsion may result in contralateral testicular alterations which appear immunologically mediated and avoidable by immunosuppression or orchiectomy of the twisted testicle within 24 hours. This study was instituted to assess three temporal aspects of these observations: (1) The effect of prepubertal torsion was studied. It was found that after prepubertal torsion, the contralateral testicle underwent normal development. (2) The duration of torsion necessary to result in contralateral testicular alterations in adult rats was studied. Deoxyribonucleic acid (DNA) histograms were utilized to assess disturbed spermatogenesis. After greater than 8 hours of torsion, detorsion offered no protection to the contralateral testicle. Marked alterations occurred in DNA histograms of 60% to 80% of the animals. (3) The duration and significance of these alterations were assessed. The alterations persisted in the contralateral testis 6 months, and the fertility rates were significantly lower than for control animals.

Prognostic significance of DNA ploidy in Ta/Tl bladder cancer: A southwest oncology group study.

While 80% of transitional cell carcinomas (TCC) present as Ta Tl lesions, they account for only 15% of deaths caused by TCC. We have evaluated the ability of DNA ploidy analysis to predict outcome in 228 patients with Ta Tl TCC. All patients were judged to be at increased risk for tumor recurrence due to having two occurrences of Stage TI tumor within 56 weeks, or three or more tumors presenting simultaneously within 16 weeks of registration. Concurrent carcinoma in situ was acceptable. All patients were treated with either bacillus Calmette Guerin (BCG) immunotherapy or mitomycin-C (MMC) intravesical chemotherapy. Patients with nondiploid tumors had higher hazard rates for both tumor progression and death (p = 0.007 and p = 0.016, respectively); however, the prognostic information of DNA ploidy was not additive to tumor grade.

False DNA aneuploidy in canine and human neoplasms.

In most cases, the appearance of aneuploid peaks in DNA histograms may be an artefact of tissue preparation or it may reflect non-stoichiometric dye binding of a cellular subpopulation rather than true DNA aneuploidy. This report reviews how false DNA aneuploidy can be recognized and eliminated from sample submitted for DNA flow cytometric analysis.

Evaluation of DNA flow cytometry as a screening test for bladder cancer.

At this present time, we feel that there is no role for DNA flow cytometry (FCM), or indeed DNA studies by any other method, to be used as a screening procedure for patients with no prior history of bladder cancer due to the high false-positive rate found when monitoring exfoliated urothelial cells. On the other hand, for patients who have had a superficial transitional cell carcinoma (TCC), which has a documented 50% recurrence rate, and depending on pathological features, a progression rate from 7 to 45%, DNA FCM provides a sensitive method to predict future disease recurrence. It provides an extremely effective way to predict future progression and further acts as a method to monitor changes in the malignant potential of the patient's disease. For those patients with a past history of superficial TCC who develop abnormal ploidy without any overt tumor, 80% will, within the next four years, suffer a disease recurrence. For the patient who has a Ta TCC and receives intravesical Bacillus Calmette-Guerin (BCG), the development of abnormal ploidy in bladder washing specimens is the single best indicator for future disease recurrence. Similarly, a negative DNA FCM of a bladder washing at six months after intravesical therapy is an excellent predictor of no further occurrence. In patients with superficial TCC, ploidy of the initial and recurrent tumor predicts for future progression. Half of those patients with stage Ta bladder cancer with two successive aneuploid bladder tumors develop muscle invasive disease within one year, while three-fourths develop advanced disease within two years after recurrence of their second aneuploid lesion.(ABSTRACT TRUNCATED AT 250 WORDS)

Human renal clear cell carcinoma: establishment and characterization of a new cell line (G-2101).

A human cell line has been established from a renal adenocarcinoma rib metastasis of a 58-y-old male. This cell line has been maintained in continuous culture for 20 mo. through more than 50 passages. It displays simultaneous expression of the intermediate filaments cytokeratin and vimentin. Flow cytometric analysis of DNA content reveals a major hyperdiploid population.