What is the restriction point?

The restriction point (R) separates two functionally different parts of G1 in continuously cycling cells. G1-pm represents the postmitotic interval of G1 that lasts from mitosis to R. G1-ps represents the pre S phase interval of G1 that lasts from R to S. G1-pm is remarkably constant in length (its duration is about three hours) in the different cell types studied so far. G1-ps, however, varies considerably, indicating that entry into S is not directly followed after passage through R. Progression through G1-pm requires continuous stimulation by mitogenic signals (e.g. growth factors) and a high rate of protein synthesis. Interruption of the mitogenic signals or moderate inhibition of protein synthesis leads to a rapid exit from the cell cycle to G0 in normal (untransformed) cells. Upon restimulation with mitogenic signals, the cell returns to the same point in G1-pm from which it left the cell cycle. Thus the cell seems to have a memory for how far it has advanced through G1-pm, suggesting that a continuous structural alteration, for example chromatin decondensation, takes place in G1. The molecular background to transition from growth factor dependence in G1-pm to growth factor independence in G1-ps (a switch which represents commitment to a new cell cycle and passage through R) is still not fully understood. Cyclin-dependent kinase (cdk)-mediated hyperphosphorylation of the retinoblastoma protein (Rb), and concomitant liberation (and activation) of members of the E2F family of transcription factors, are probably important aspects of R control in normal cells. A key component here could be cdk2 activity which is controlled by cyclin E. When cdk2 activity starts to increase rapidly in G1, due to activation of a positive feedback loop, it reaches a critical level above which cdk inhibitors (CKIs) such as p21 and p27 are outweighed; the cell has then become independent of mitogenic and inhibitory signals and is committed to a new cell cycle. However, other components are probably also involved in R control. For instance, a 'cryptic' R (a G1-pm-like state) can be induced even in tumour cells that do not respond to growth factor starvation or protein synthesis inhibitors, and are therefore probably defective in the cdk-Rb-E2F pathway. Possibly, a certain degree of chromatin decondensation has to take place after mitosis in order to allow transcription of, for example, the cyclin E gene or other critical E2F targets. Although the molecular basis for restriction point control still remains unclear, we can expect rapid progress in this important field over the next few years.

Genomic instability and proliferative activity as risk factors for distant metastases in breast cancer.

The role of genomic instability and proliferative activity for development of distant metastases in breast cancer was analysed, and the relative contribution of these two risk factors was quantified. A detailed quantitative comparison was performed between Ki67 and cyclin A as proliferative markers. The frequency of Ki67 and cyclin A-positive cells was scored in the same microscopic areas in 428 breast tumours. The frequency of Ki67-positive cells was found to be highly correlated with the frequency of cyclin A-positive cells, and both proliferation markers were equally good to predict risk of distant metastases. The relative contribution of degree of aneuploidy and proliferative activity as risk markers for developing distant metastases was studied independently. Although increased proliferative activity in general was associated with an increased risk of developing distant metastases, ploidy level was found to be an independent and even stronger marker when considering the group of small (T1) node negative tumours. By combining proliferative activity and ploidy level, a large group of low risk breast tumours (39%) could be identified in which only a few percentage of the tumours (5%) developed distant metastases during the 9-year follow-up time period.

Accumulation of cyclin E is not a prerequisite for passage through the restriction point.

The restriction point (R) is defined as the point in G(1) after which cells can complete a division cycle without growth factors and divides G(1) into two physiologically different intervals in cycling cells, G(1)-pm (a postmitotic interval with a constant length of 3 to 4 h) and G(1)-ps (a pre-DNA-synthetic interval with a variable length of 1 to 10 h). Cyclin E is a G(1) regulatory protein whose accumulation has been suggested to be critical for passage through R. We have studied cyclin E protein levels in individual cells of asynchronously growing cell populations, with respect to both passage through R and entry into S phase. We found that the postmitotic G(1) cells that had not yet reached R were negative for cyclin E accumulation. On the other hand, cells that had passed R were found to accumulate cyclin E at variable times (1 to 8 h) after passage through R and 2 to 5 h before entry into S. These kinetic data rule out the hypothesis that passage through R is dependent on the accumulation of cyclin E but suggest, instead, the converse, that passage through R is a prerequisite for cyclin E accumulation. Furthermore, we found that most of the cyclin E protein is downregulated within 1 to 2 h after entry into S.

Kinetics of cell replication of the uterine cervix. III. Delayed migration in atypical epithelia of mice.

The migration of tritiated thymidine ([3H]dThd)-labeled cells toward the epithelial surface was recorded at various intervals (from 1 to 240 hr) in the normal and atypical cervical epithelial of 127 C57BL mice. Cervical atypias were induced by local application of 3,4-benzo[a]pyrene for 5 months9 This treatment was discontinued 7 days before the animals were killed. Cells were labeled by a single ip injection of 6 muCi [3H]dThd/g body weight. The disappearance of heavily labeled cells in normal animals 96 hours after the pulse and in solvent-treated controls after 144 hours suggested that the original labeled cells had been exfoliated. However, cervical atypias showed heavily labeled cells as much as 240 hours after the [3H]dThd incorporation. Universal epithelial labeling (suggesting repopulation by daughter cells) usually occurred 24--48 hours after pulse labeling in normal and control animals but occurred after 96--240 hours in animals with atypical epithelium. On the basis of these results, the pace of both cellular intraepithelium transmigration and of epithelial repopulation by atypical daughter cells is substantially retarded during the early stages of cervical carcinogenesis.

Kinetics of G1 progression in 3T6 and SV-3T3 cells following treatment by 25-hydroxycholesterol.

The kinetics of cell cycle progression in continuously proliferating 3T3 fibroblasts and two tumor transformed derivatives (3T6 and SV 3T3 cells) following treatment by growth-factor deprivation (serum starvation) or 25-hydroxycholesterol were studied. Normal 3T3 cells were found to respond immediately (in the first cycle) to growth factor deprivation by leaving the cell cycle from G1, whereas the tumor transformed derivatives did not. However, all three cell types were forced to stop the progression through the beginning of G1 when treated by 25-hydroxycholesterol. It was ensured that the doses of 25-hydroxycholesterol used really induced substantial decrease of HMG CoA reductase activity. However, the effects of serum starvation on HMG CoA reductase activity varied considerably. In 3T3 cells HMG CoA reductase activity was substantially depressed, in 3T6 cells it was moderately depressed, and in SV-3T3 cells it was not depressed at all. This difference of HMG CoA reductase activity between 3T6 and SV-3T3 cells was related to the difference of growth activity in serum-free medium. The data indicate that a certain activity of HMG CoA reductase is required for the proliferation of normal as well as tumor transformed cells but also that impairment of the control of HMG CoA reductase, leading to increased enzyme activity, may result in uncontrolled growth in tumor transformed cells.

Ploidy level determinations in high-grade and low-grade malignant variants of prostatic carcinoma.

Ploidy level determinations of prostatic carcinoma in 213 patients selected on the basis of survival time were analyzed. Two extreme groups were chosen: extreme group 1, 131 patients who died from prostatic cancer within 3 years of diagnosis; and extreme group 2, 82 patients who survived for more than 15 years after diagnosis. All patients were diagnosed by fine needle aspiration biopsy. The DNA measurements were performed on Feulgen stained, destained May-Grünwald-Giemsa smears. The DNA distribution patterns were studied in three benign prostatic lesions as a base for analyses of prostatic carcinoma. By choosing two extreme groups it was possible to evaluate in detail the optimal limits for defining the diploid 2c region and the tetraploid 4c region. Various limits were tested in order to determine those which most clearly separate extreme group 1 from extreme group 2. We found that the optimal upper diploid limit was 2.5c and the optimal tetraploid limits were 3.5c-4.5c. By using these limits to determine the percentage of aberrant tumor cells, i.e., non-2c and non-4c, and combining this with the modal value (in c units) of the tumor cell population the tumors could unambiguously be divided into D-, T-, and A-types. D/T-type tumors were found only in extreme group 2 and had less than 30% aberrant tumor cells, while A-type tumors (high-grade aneuploid) had greater than 50% non-2c and non-4c tumor cells. All A-type tumors were found in extreme group 1. In order to investigate whether the classification of tumors into D-, T-, and A-type was valid in general and could also be applied to patients with survival time between the two extreme groups (3-15 years), a material of 79 patients with a wide range of survival times was tested. The tumors were classified according to the above-mentioned criteria into A-, D-, and T-type tumors. All patients who died within 5 years of diagnosis had A-type tumors. All patients who lived greater than 5 years from diagnosis had D- or T-type tumors.

Cytotoxic effects of cell cycle phase specific agents: result of cell cycle perturbation.

Although agents which act in a cell cycle phase specific manner are commonly used in the clinic and in basic research, it is as yet unclear why these agents are cytotoxic. In this paper, we examine the cellular events associated with the cytotoxicity of aphidicolin and vincristine in CHO strain AA8 cells. Cell killing resulting from aphidicolin treatment was found to require a period of inhibition-free growth following removal of the drug and was associated with characteristic aberrant mitotic processes. The cytotoxic effects of aphidicolin could be antagonized by the concomitant inhibition of protein synthesis with cycloheximide in the period of DNA synthesis inhibition. Cell killing resulting from treatment with vincristine was associated with the aberrant segregation of nuclear material and the formation of multiple partial nuclei. Vincristine cytotoxicity was found to be antagonized by concomitant administration of cycloheximide or cytochalasin D. These data support a hypothesis that the cytotoxic effects of cell cycle phase specific agents do not derive directly from their biochemical actions per se. We propose that cell death results from processes that are evoked by dissociation of normally integrated cell cycle events, and that dissociation involves replicative/mitotic events in the case of aphidicolin and karyokinetic/nuclear reformation events in the case of vincristine.

Abolition of mevinolin-induced growth inhibition in human fibroblasts following transformation by simian virus 40.

The basal level of the gene expression and the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase was higher in SV40-transformed human fibroblasts (90-VA VI) than in normal ones (HDF). In both these cell types mevinolin (25 microM) caused an 85-90% depression of HMG CoA reductase activity and of the incorporation of [3H]acetate into sterols. In HDF this was coupled to an efficient block of cell growth, whereas the growth of 90-VA VI was only slightly reduced by mevinolin. In HDF, mevinolin (25 microM) also abolished essentially all dilichol synthesis, as measured by incorporation of [3H]acetate. In contrast, dolichol synthesis remained unaltered, or was increased, in mevinolin-treated 90-VA VI. We suggest that these different responses of dolichol synthesis may depend on different substrate affinities of the rate-limiting enzyme in the dolichol pathway. However, if 90-VA VI was treated with 25-hydroxycholesterol (25-OH), an alternative inhibitor of HMG CoA reductase, the cellular growth as well as dolichol synthesis was significantly decreased. Since the inhibitory effect of 25OH on HMG CoA reductase activity did not exceed that of mevinolin, it seems that 25-OH, besides HMG CoA reductase, inhibits steps distal to HMG CoA reductase. This notion was further supported by the finding that addition of mevalonate did not prevent the 25-OH-induced growth inhibition. However, if dolichol was added along with 25-OH, the block was partially prevented, indicating that a critical level of de novo synthesis of dolichol for cellular growth.

Correlation between clinical response to hormone therapy and steroid receptor content in prostatic cancer.

Hormonal therapy is the dominating form of treatment for prostatic carcinoma. The majority of cases (80%) are well controlled for varying times with this regimen. However, thus far there have been no adequate methods to predict in which cases hormonal therapy is of less benefit. Measurement of cancer tissue content of intracellular hormone receptors constitutes progress toward a more individualized therapy in prostatic carcinoma. In this study biopsies from 16 cancer patients were taken before therapy was given, and the specimens were analyzed with regard to content of specific methyltrienolone-binding sites. A correlation has been made between receptor content and clinical response to hormonal therapy in each case. Twelve specimens contained measurable amounts of steroid receptors. Of these, one patient died during irradiation therapy before onset of hormonal treatment. However, of the remaining 11 patients, 9 responded well to hormones (9/11 approximately 82%). The two receptor-positive nonresponders had the lowest measurable receptor levels in the series. Four specimens contained no detectable amounts of receptors. Three of these patients showed no response to therapy (3/4 = 75%) but one was "false negative." Our data indicate that steroid receptor analysis may become a valuable diagnostic tool in individualizing the therapy for prostatic cancer.

Prognostic significance of nuclear DNA content in serous ovarian tumors.

Specimens from 73 serous ovarian cancers were examined with respect to DNA content of the tumor cells. The prognostic value of DNA analysis, as reflected in patient survival, was retrospectively compared with that of conventional histological assessment of cancer. DNA in individual tumor cells was measured in sections from the original paraffin-embedded specimens. High proportions of cells with very high DNA values were identified in tumors from 16 patients, 10 of whom died of the disease during the follow-up period. The histological classification was invasive adenocarcinoma in 47 tumors and borderline cancer in 26. All of the patients who died had invasive adenocarcinoma. Although both DNA analysis and histological evaluation were sensitive predictors of mortality, the specificity of the DNA method was distinctly higher (0.90 versus 0.42). The study suggests that analysis of the DNA content of tumor cells can be a useful supplement to histological assessment of cancer and accordingly can significantly assist in the planning of treatment.

Comparative genomic hybridization of formalin-fixed, paraffin-embedded breast tumors reveals different patterns of chromosomal gains and losses in fibroadenomas and diploid and aneuploid carcinomas.

Comparative genomic hybridization serves as a screening test for regions of copy number changes in tumor genomes. We have applied the technique to map DNA gains and losses in 33 cases of formalin-fixed, paraffin-embedded primary breast tumors (13 fibroadenomas and 10 diploid and 10 aneuploid carcinomas). No genomic imbalances were found in fibroadenomas. Recurrent findings in adenocarcinomas include copy number increases for chromosomes 1q (14 of 20 samples), 8q (10 of 20), 17q (5 of 20), 6p (3 of 20), 13q (3 of 20), and 16p (3 of 20), and copy number decreases for chromosomes 22 (7 of 20), 17p (6 of 20), and 20 (3 of 20). Regional high level copy number increases were observed on chromosome bands 1q32, 8p11, 8q24, 10p, 11q13, 12p, 12q15, 17q11-12, and 17q22-24. The majority of the samples were studied for gene amplification of c-myc, c-erbB2, cycD1, and int-2 by means of Southern blot analysis. The comparison with DNA ploidy measurements revealed a different distribution and a significantly higher number of chromosomal aberrations in aneuploid tumors than in diploid tumors and in fibroadenomas.

Comparison of nuclear DNA content in primary and metastatic papillary thyroid carcinoma.

Nuclear DNA values were determined in 40 primary papillary thyroid carcinomas, as well as in 52 corresponding local recurrences and metastases were observed either at the time of diagnosis or up to 20 years later. The patient population consisted of 34 survivors and 6 nonsurvivors. In survivors, both the primary tumors and their recurrences and metastases exhibited a majority of cells with DNA values within the normal diploid region, whereas nonsurvivors showed increased and scattered DNA values. In all cases, the primary tumors and the corresponding recurrences and metastases showed similar DNA distribution patterns even if many years had passed between the detection of the primary tumor and the metastases. The results indicate that in papillary thyroid carcinomas, the DNA distribution patterns in the primary tumor and the corresponding recurrences or metastases are generally similar throughout the entire period of disease.

Frequent amplification of the telomerase reverse transcriptase gene in human tumors.

Activation of telomerase is a crucial step during cellular immortalization and malignant transformation of human cells and requires the induction of the catalytic component, human telomerase reverse transcriptase (hTERT), encoded by the hTERT gene. It is poorly understood how the hTERT gene is activated in human cancer cells. In the present study, we examined the hTERT gene copy number in human cancer cell lines and in primary tumor tissues. Amplification of the hTERT gene was observed in 8 of 26 (31%) tumor cell lines and 17 of 58 (30%) primary tumors examined (8 of 21 lung tumors, 3 of 10 cervical tumors, 5 of 19 breast carcinomas, and 1 of 8 neuroblastomas). In addition, 13 of 26 (50%) cell lines and 13 of 58 (22%) primary tumors displayed gain of hTERT gene copies with 3-4 copies/cell. The present findings imply that the hTERT locus may be a frequent target for amplification during tumorigenesis and that this genetic event probably contributes to the dysregulation of telomerase activity occurring in human tumors.

Existence of a commitment program for mitosis in early G1 in tumour cells.

The proliferation of normal non-tumourigenic mouse fibroblasts is stringently controlled by regulatory mechanisms located in the postmitotic stage of G1 (which we have designated G1pm). Upon exposure to growth factor depletion or a lowered de novo protein synthesis, the normal cells leave the cell cycle from G1pm and enter G0. The G1 pm phase is characterized by a remarkably constant length (the duration of which is 3 h in Swiss 3T3 cells), whereas the intercellular variability of intermitotic time is mainly ascribable to late G1 or pre S phase (G1ps) (Zetterberg & Larsson (1985) Proc. Natl. Acad. Sci. USA 82, 5365). As shown in the present study two tumour-transformed derivatives of mouse fibroblasts, i.e. BPA31 and SVA31, did not respond at all, or only responded partially, respectively, to serum depletion and inhibition of protein synthesis. If the tumour cells instead were subjected to 25-hydroxycholesterol (an inhibitor of 3-hydroxy-3 methyglutaryl coenzyme A reductase activity), their growth was blocked as measured by growth curves and [3H]-thymidine uptake. Time-lapse analysis revealed that the cells were blocked specifically in early G1 (3-4 h after mitosis), and DNA cytometry confirmed that the arrested cells contained a G1 amount of DNA. Closer kinetic analysis revealed that the duration of the postmitotic phase containing cells responsive to 25-hydroxycholesterol was constant. These data suggest that transformed 3T3 cells also contain a 'G1pm program', which has to be completed before commitment to mitosis. By repeating the experiments on a large number of tumour-transformed cells, including human carcinoma cells and glioma cells, it was demonstrated that all of them possessed a G1pm-like stage. Our conclusion is that G1pm is a general phenomenon in mammalian cells, independent of whether the cells are normal or neoplastic.

Reliability and significance of DNA measurements in interphase nuclei and division figures in histological sections.

DNA contents from single cells at interphase and division were analysed in histological sections and in imprints from 73 breast cancer specimens. Fetal livers from 18 terminations of normal pregnancies provided the standard for truly mitotic prophases, metaphases and telophases. The reliability of DNA quantities from image microphotometry was improved using paraffin-embedded tissue samples from which 4, 8 and 15 microns slices were Feulgen stained. Imprinted replicas from the mirror surface of each freshly cut specimen provided matching domains and represent the crucial approach in this project. A close positive relationship was observed between interphase nuclei in 8 microns sections and their imprinted counterparts (r = 0.992; n = 73). Interphase nuclei in 4 microns sections yielded insufficient DNA contents when compared with the imprints (r = 0.815; n = 21) and with endogenous lymphocyte nuclei. This 2 cDNA standard also calibrated 232 mitotic figures to 3.91 +/- 0.01 c in 15 microns sections from fetal liver. Prophases, metaphases and telophases were slightly scattered (coefficient of variation = 0.04 each). The 0.09 c deficiency to plain 4.0 c was read as an artifact from sectioning. However, the methodical bias did not challenge the most irregular DNA distribution profiles recorded from chromosome division figures (CDFs) in 15 microns sections of breast cancers. Poorly differentiated and aggressive breast cancer (Auer type IV, Zetterberg type A) exhibited a 4.5 c exceeding rate of 82.24% from a total of 752 CDFs in 10 randomly selected cases. Well differentiated, slowly growing cancer with diploid interphase nuclei (Auer I, Zetterberg D) surprisingly showed a 4.5 c exceeding rate of 29.26% from a total of 173 mitoses and CDFs in 10 randomly selected cases. The bulk of data beyond the mitotic 4.0 c level discriminates biological bias from methodical impairment. We concluded that 8 microns sections are sufficient for human interphase nuclei, whereas a depth of 15 microns preserves intact mitoses and CDFs.

Detection of TP53 gene mutations in human sarcomas.

To determine the frequency and type of TP53 mutations in human sarcomas, we examined exons 5-8 of the TP53 gene in 48 sarcomas using single-strand conformation polymorphism (SSCP) analysis and direct sequencing. Nine tumours had mobility-shifts on SSCP analysis, and sequencing of six of these tumours revealed 10 mutations: one insertion, two deletions and seven point mutations (four transitions and three transversions). Four of these mutations resulted in frame-shifts, one in a truncated protein, four cases in mono-allelic point mutations and one case in an altered splice site. These data show that approximately 20% of sarcomas harbour TP53 gene alterations and illustrate a variety of TP53 gene mutation types.

Relationship between oncogene amplification, aneuploidy and altered expression of p53 in breast cancer.

Many studies have noted an association between amplification of single oncogenes and poor prognosis in breast cancer. We are investigating whether measurement of amplification in a larger number of proto-oncogenes increases the reliability of the prognostic information provided. As the first stage of this investigation, amplification (of c-erbB-2, cycD1, int-2, c-myc and MDM2), aneuploidy and altered expression of p53, which all indicate genetic instability, were studied in 117 primary breast adenocarcinomas. Amplification was correlated with aneuploidy (p=0.002) but not with altered expression of p53 even though the tumours with p53 overexpression were all aneuploid. Our results suggest that measurement of amplification is a potentially valuable prognostic factor.

Immunohistochemical expression of the mutant p53 protein and nuclear DNA content during the transition from benign to malignant breast disease.

Immunohistochemical expression of the cellular phosphoprotein p53 was investigated in archival, formalin-fixed, and paraffin-embedded surgical breast tissue specimens from 543 patients using the polyclonal antibody CM-1. Cytometric DNA assessments were performed on histopathologically or cytopathologically identified cell nuclei using image analysis. The series included five samples of normal resting breast parenchyma, 35 benign lesions including benign tumors, 54 hyperplastic lesions with and without atypia, 109 carcinomas in situ, and 340 invasive adenocarcinomas. In 56 of the latter cases specimens from corresponding lymph node metastases also were investigated. Mutant p53 protein expression was absent in normal resting parenchyma and in benign lesions, including benign tumors and epithelial hyperplasias. However, 14 of the 54 hyperplasias (26%) were found to be of DNA aneuploid type. Thirteen of 109 (12%) carcinomas in situ and 79 of 340 (23%) invasive neoplasms expressed the mutant p53 protein. Eight of nine (89%) p53 immunoreactive carcinomas in situ and 62 of 78 (80%) invasive carcinomas with p53 expression were DNA aneuploid. In invasive carcinomas p53 expression was absent in well differentiate neoplasms. In contrast, 58 of 158 (37%) poorly differentiated invasive carcinomas immuoreacted. Intraductal carcinomas of comedo type and poorly differentiated invasive carcinomas of comedo type expressed the mutant p53 protein in seven of 18 cases (39%) and in 14 of 22 cases (64%), respectively. The staining behavior of lymph node metastases was the same as that of the corresponding primary tumors. The present findings suggest that chromosomal alterations as indicated by DNA aneuploidy occur in precancerous lesions.(ABSTRACT TRUNCATED AT 250 WORDS)

The relationship between aneuploidy and p53 overexpression during genesis of colorectal adenocarcinoma.

This paper describes the investigation of nuclear DNA content and p53 immunoreactivity in normal mucosa (n = 25), mildly (n = 15), moderately (n = 28) and severely atypical (n = 22) colorectal adenomas and in colorectal adenocarcinomas (n = 116). Twenty-seven per cent of the mildly atypical, 43% of the moderately, 77% of the severely atypical adenomas and 91% of the colorectal carcinomas were distinctly aneuploid. In the aneuploid lesions p53 immunoreactivity was not observed in mildly atypical adenomas, whereas 17% of the moderately atypical, 24% of the severely atypical adenomas and 66% of the adenocarcinomas were p53 positive. None of the diploid lesions were p53 immunoreactive. These data are interpreted to indicate that genomic instability as reflected by crude aneuploidy occurs early during genesis of colorectal carcinoma and represents a high risk factor for p53-gene mutation.