Disruption of p53 in human cancer cells alters the responses to therapeutic agents.

We have examined the effects of commonly used chemotherapeutic agents on human colon cancer cell lines in which the p53 pathway has been specifically disrupted by targeted homologous recombination. We found that p53 had profound effects on drug responses, and these effects varied dramatically depending on the drug. The p53-deficient cells were sensitized to the effects of DNA-damaging agents as a result of the failure to induce expression of the cyclin-dependent kinase inhibitor p21. In contrast, p53 disruption rendered cells strikingly resistant to the effects of the antimetabolite 5-fluorouracil (5-FU), the mainstay of adjuvant therapy for colorectal cancer. The effects on 5-FU sensitivity were observed both in vitro and in vivo, were independent of p21, and appeared to be the result of perturbations in RNA, rather than DNA, metabolism. These results have significant implications for future efforts to maximize therapeutic efficacy in patients with defined genetic alterations.

Requirement for p53 and p21 to sustain G2 arrest after DNA damage.

After DNA damage, many cells appear to enter a sustained arrest in the G2 phase of the cell cycle. It is shown here that this arrest could be sustained only when p53 was present in the cell and capable of transcriptionally activating the cyclin-dependent kinase inhibitor p21. After disruption of either the p53 or the p21 gene, gamma radiated cells progressed into mitosis and exhibited a G2 DNA content only because of a failure of cytokinesis. Thus, p53 and p21 appear to be essential for maintaining the G2 checkpoint in human cells.

Menopause: when hormone replacement therapy is not an option. Part I.

Although the perimenopausal period is often experienced as a positive life transition, it is frequently accompanied by a variety of distressing physical and emotional sequelae. Hormone replacement therapy (HRT) has been hailed as the first-line treatment for many of these symptoms. A significant number of women, however, are unable to take exogenous hormones because of absolute or relative contraindications to therapy. Other women are unwilling to use this treatment for a variety of reasons, including reluctance to use unnatural exogenous hormones and fear of unknown risks of HRT. This two-part review discusses the physiology of menopause and its related symptoms, as well as the risks and benefits of both oral and non-oral routes of hormone administration. Self-help measures and alternative therapeutic options are recommended for the treatment of menopausal symptoms, which include vasomotor instability, urogenital atrophy, psychologic disturbances, and risk of osteoporosis and cardiovascular disease.

Cell-cycle arrest versus cell death in cancer therapy.

In response to anticancer therapeutics, human colon cancer cells growing in vitro either enter into a stable arrest or die, depending on the integrity of their cell-cycle checkpoints. To test whether altered checkpoints can modulate sensitivity to treatment in vivo, xenografts were established from isogenic lines differing only in their p21 checkpoint status. Although all tumors with intact checkpoint function underwent regrowth after treatment with gamma-radiation, a significant fraction of checkpoint-deficient tumors were completely cured. This difference in sensitivity was not detected by the clonogenic survival assay, because both arrest and death preclude outgrowth of colonies. These results demonstrate that checkpoint status affects sensitivity to anticancer treatments in vivo, and these findings have important implications for identifying and testing new therapeutic compounds.

The adult personality of childhood incest victims.

This is a further report on a group of 30 incest survivors we wrote about in 1994. Here, we report on measures of personality derived from the Apperceptive Personality Test, which contains many variables similar to those on the one we reported in 1994. On both measures, incest survivors can be characterized as having more negative perceptions and self-descriptions than the comparison group.

Genetic determinants of p53-induced apoptosis and growth arrest.

Previous studies have suggested that expression of p53 in cancer cells can result in either growth arrest or apoptosis. Accordingly, expression of p53 in a series of colorectal cancer cell lines yielded growth arrest in some lines (A-lines) and apoptosis in others (D-lines). To investigate the basis of this difference, we evaluated the role of p21WAF1/Cip1, a known mediator of p53-induced growth arrest. Inactivation of p21 by homologous recombination converted an A-line to a D-line, suggesting that p21 could protect cells from apoptosis. However, examination of p53-induced p21 expression in naturally occurring D-lines and A-lines demonstrated that the induction of p21 could not account for the differential response to p53. Moreover, when a D-line was fused to an A-line, the resulting hybrid cells underwent apoptosis in response to p53, indicating that the apoptosis pathway was dominant over the growth arrest pathway. Therefore, the apoptotic response to p53 in colorectal cancer cells is modulated by at least two factors: p21-mediated growth arrest that can protect cells from apoptosis in A-cells, and trans-acting factors in D-cells that can overcome this protection, resulting in cell death.

Suppression of cancer cell growth by adenovirus expressing p21(WAF1/CIP1) deficient in PCNA interaction.

p53 tumor suppression is deficient in the majority of human cancers. Efforts to understand this pathway have identified cyclin-dependent kinase (CDK) inhibitors and suggested a potential for their replacement in human cancer. In the present studies, expression of a C-terminal deletion mutant of the human p21(WAF1/CIP1) CDK inhibitor completely suppressed the growth of colon cancer cells, whereas full-length p21 only partially suppressed growth. We prepared a replication-deficient adenoviral recombinant which expresses the p21 C-terminal mutant (Ad-WAF1-341) and compared its tumor suppressive abilities with Ad-p53 and Ad-LacZ. Ad-WAF1-341- and Ad-p53-infected cancer cells, but not Ad-LacZ-infected cancer cells, expressed a nuclear protein recognized by anti-p21 antibody and were deficient in cell cycle progression. The exogenous p21 mutant interacted with CDK2 but not proliferating cell nuclear antigen following infection of p21-/- cancer cells. Ad-WAF1-341 was more potent than Ad-p53 in inhibiting DNA synthesis in human papillomavirus 16 E6-expressing cancer cells. Most importantly, the Ad-WAF1-341-infected E6-expressing cells died, whereas most of the Ad-p53-infected cells continued to proliferate. Endonucleolytic cleavage of DNA was observed in Ad-WAF1-341-infected cancer cells. These observations suggest that Ad-WAF1-341 should be evaluated in the treatment of human papillomavirus-associated neoplasia and other neoplasias resistant to p53.

Uncoupling of S phase and mitosis induced by anticancer agents in cells lacking p21.

Precise coordination of the S and M phases of the eukaryotic cell cycle is critical not only for normal cell division, but also for effective growth arrest under conditions of stress. When damaged, a cell must communicate signals to both the mitotic and DNA synthesis machineries so that a mitotic block is not followed by an extra S phase, or vice versa. The biochemical mechanisms regulating this coordination, termed checkpoints, have been identified in lower eukaryotes, but are largely unknown in mammalian cells. Here we show that p21 WAF1/CIP1, the prototype inhibitor of cyclin-dependent kinases (CDKs), is required for this coordination in human cells. In the absence of p21, DNA-damaged cells arrest in a G2-like state, but then undergo additional S phases without intervening normal mitoses. They thereby acquire grossly deformed, polyploid nuclei and subsequently die through apoptosis. Perhaps not by coincidence, the DNA-damaging agents that can cause S/M uncoupling are used in the clinic to kill cancer cells preferentially.

Repair Defect in p21 WAF1/CIP1 -/- human cancer cells.

p53 induction and cell cycle arrest occur following DNA damage, possibly to allow repair prior to replication. p21WAF1/CIP1, a cyclin-cyclin-dependent kinase inhibitor and proliferating cell nuclear antigen-interacting protein, is induced by p53 and mediates the cell cycle arrest. To investigate a role for p21 in DNA repair in vivo, we studied the expression of in vitro damaged reporter DNA transfected into p21 +/+ or -/- HCT116 human colon cancer cells. Introduction of UV-damaged or cisplatinum-damaged cytomegalovirus-driven beta-galactosidase reporter DNA into tumor cells revealed a significant decrease (2-5-fold) in reporter expression in p21 -/- versus +/+ cells. In the absence of DNA damage, there was a significant increase (2-3-fold) in the number of 6-TG-resistant colonies derived from p21 -/- versus +/+ cells. Reintroduction of wild-type p21, but not a p21 C-terminal truncation mutant which lacks the proliferating cell nuclear antigen interaction domain, stimulated (2-3-fold) the repair capacity of the p21-deficient cells. We conclude that p21 deficiency is associated with a defect in DNA repair, which could lead to an increased sensitivity of tumor cells to DNA damage.

p21 is necessary for the p53-mediated G1 arrest in human cancer cells.

DNA-damaging agents induce a p53-dependent G1 arrest that may be critical for p53-mediated tumor suppression. It has been suggested that p21WAF1/CIP1, a cdk inhibitory protein transcriptionally regulated by p53, is an effector of this arrest. To test this hypothesis, an isogenic set of human colon adenocarcinoma cell lines differing only in their p21 status was created. The parental cell line underwent the expected cell cycle changes upon induction of p53 expression by DNA damage, but the G1 arrest was completely abrogated in p21-deficient cells. These results unambiguously establish p21 as a critical mediator of one well-documented p53 function and have important implications for understanding cell cycle checkpoints and the mechanism(s) through which p53 inhibits human neoplasia.

Topological control of p21WAF1/CIP1 expression in normal and neoplastic tissues.

The p53-regulated gene product p21WAF1/CIP1 is the prototype of a family of small proteins that negatively regulate the cell cycle. To learn more about p21WAF1/CIP1 regulation in vivo, monoclonal antibodies were developed for immunohistochemistry. These revealed that p21WAF1/CIP1 expression followed radiation-induced DNA damage in human skin in a pattern consistent with its regulation by p53. A detailed comparison of the human, rat, and mouse p21WAF1/CIP1 promoter sequences revealed that this induction was probably mediated by conserved p53-binding sites upstream of the transcription start site. In unirradiated tissues, p21WAF1/CIP1 expression was apparently independent of p53 and was observed in a variety of cell types. Moreover, there was a striking compartmentalization of p21WAF1/CIP1 expression throughout the gastrointestinal tract that correlated with proliferation rather than differentiation. As epithelial cells migrated up the crypts, the Ki67-expressing proliferating compartment near the crypt base ended abruptly, with the coincident appearance of a nonproliferating compartment expressing p21WAF1/CIP1. In colonic neoplasms, this distinct compartmentalization was largely abrogated. Cell cycle inhibitors are thus subject to precise topological control, and escape from this regulation may be a critical feature of neoplastic transformation.

p53 tagged sites from human genomic DNA.

The product of the tumor suppressor gene p53 binds to DNA and activates transcription from promoters containing its consensus binding site. This activity has been hypothesized to be responsible for its biological effects. However, the total number and nature of human genomic sites with which p53 can functionally interact is unknown. In this paper, we have used a Saccharomyces cerevisiae-based screen to identify human genomic sequences that activate transcription from an adjacent reporter gene in a p53-dependent manner (p53-tagged sites, PTS). Fifty-seven different PTS were identified, and the total number of such sites in the human genome was predicted to be between 200 and 300. Almost all contained two adjacent copies of the previously defined consensus 5'-PuPuPuC(A/T)(T/A)GPyPyPy-3'. Spacing between the copies was found to be critical for sequence-specific transcriptional activation in vivo. These results further refine the nature of the genomic sequences likely to be most important for p53-mediated tumor suppression.

Personality characteristics of incest survivors on the Draw-A-Person Questionnaire.

We investigated the psychological sequelae of incest through the use of the Draw-A-Person Questionnaire (DAPQ; Karp, 1990), a projective technique with an objective component. A group of adult incest survivors (n = 30) and a matched control sample (n = 30) participated in our study. Ratings of characters drawn by subjects were compared between the two groups on 10 measures hypothesized to differentiate the groups. Results suggest that incest survivors project more negative characteristics on their drawn characters than do control women. In addition, survivors treat their male and female characters differently to a greater extent than do controls. Findings indicate that the DAPQ is a valuable method to detect long-lasting repercussions that may stem from an incestuous experience. Specific differences between the groups are discussed, as well as suggestions for future research.

Recombinant toxins containing the variable domains of the anti-Tac monoclonal antibody to the interleukin-2 receptor kill malignant cells from patients with chronic lymphocytic leukemia.

We have previously shown that the variable domains of the monoclonal antibody anti-Tac [anti-Tac(Fv)] can be fused to derivatives of Pseudomonas exotoxin (PE) or diphtheria toxin (DT) to produce recombinant immunotoxins that kill interleukin-2 (IL-2) receptor-bearing cells. We now report that two of these single-chain recombinant immunotoxins, anti-Tac(Fv)-PE40KDEL and DT388-anti-Tac(Fv), are cytotoxic toward peripheral blood mononuclear cells (PBMCs) from patients with chronic lymphocytic leukemia (CLL). In anti-Tac(Fv)-PE40KDEL, anti-Tac(Fv) is genetically fused to the amino terminus of PE40KDEL, a recombinant form of PE which contains amino acids 253-608 of PE and the -KDEL mutation at the carboxyl terminus. In DT388-anti-Tac(Fv), anti-Tac(Fv) is fused to the carboxyl terminus of the first 388 amino acids of DT. PBMCs from 14 patients were incubated with the recombinant toxins for 60 hours, and [3H]-leucine incorporation was measured. Anti-Tac(Fv)-PE40KDEL was cytotoxic to 7 of the 14 patient samples, with half-maximal inhibition of protein synthesis (IC50) achieved at 1.2 to 9 ng/mL (1.8 to 13 x 10(-11) mol/L). DT388-anti-Tac(Fv) was cytotoxic to 11 of the 14 samples, with IC50s ranging from less than 1 to 250 ng/mL. DT388-IL-2, in which the first 388 amino acids of DT are attached to IL-2, was marginally cytotoxic toward only 4 of 13 CLL samples tested with IC50s ranging from 100 to 550 ng/mL. Trypan blue staining of cells from several patients indicated that inhibition of protein synthesis correlated with cell death. Binding assays using [3H]-anti-Tac indicated that the CLL cells from nine of the patients contained between 400 and 2,500 sites per cell. Cells from another patient, which were resistant to both anti-Tac(Fv)-PE40KDEL and DT388-anti-Tac(Fv), had less than 100 sites per cell. We conclude that anti-Tac(Fv)-PE40KDEL and DT388-anti-Tac(Fv) can kill CLL cells which have low numbers of IL-2 receptors, and should be investigated further for therapy of this disease.

Human T-cell gamma chain genes: organization, diversity, and rearrangement.

The human T-cell gamma chain genes have been characterized in an attempt to better understand their role in immune response. These immunoglobulin-like genes are encoded in the genome in variable, joining, and constant segments. The human gamma genes include at least six variable region genes, two joining segments, and two constant-region genes in germline DNA. Variable and joining segments recombine during the development of T cells to form rearranged genes. The diversity of human gamma genes produced by this recombinational mechanism is greater than that produced by the murine genome but is more limited than that of other immunoglobulin-like genes.

Association of human T-cell leukaemia/lymphoma virus with the Tac antigen marker for the human T-cell growth factor receptor.

Certain adult T-cell lymphoproliferative disorders are associated with human T-cell leukaemia virus (HTLV), a unique human type C retrovirus. (The strains of HTLV used in these studies belong to the subgroup HTLV-I.) HTLV is not an endogenous agent in man, but rather is an acquired virus with T-cell tropism. Neoplastic cells from patients infected with HTLV generally express receptors for T-cell growth factor (TCGF) (interleukin-2), and do not require prior activation with antigens or lectins to undergo TCGF-induced proliferation. Furthermore, neoplastic T-cell lines originating from such patients may constitutively produce TCGF, TCGF receptors and HTLV virions. HTLV is transmissible from cell to cell, and the infection of human T cells in vitro is associated with the expression of TCGF receptors, which can be identified by the monoclonal antibody termed anti-Tac. In our experience to date, T-cell populations that produce HTLV without exception also express epitopes found on TCGF receptors. Recognition of an association between HTLV virions and the Tac antigen would have clinical and theoretical implications. We now present evidence that during the replication or release of HTLV, the virion becomes preferentially associated with the Tac antigen.

Amniotic fluid alpha-fetoprotein as a marker in prenatal diagnosis of neural tube defects.

The prenatal diagnosis of anencephaly and spina bifida (neural tube defect, NTD) through amniotic fluid analysis for alpha-fetoprotein (AFP) is gradually gaining clinical recognition. AFP concentrations were determined in 237 amniotic fluids from normal pregnancies ranging between 7 and 42 weeks of gestation. A steady decline in AFP from 26 mug/ml at 7-9 weeks to 155 ng/ml at term is observed. AFP concentration was determined in 35 amniotic fluids from 33 confirmed neural tube defective pregnancies. In 14 cases where amniotic fluid was examined prior to the 26th week of gestation. AFP was markedly elevated when compared with the normal range of the same gestational period. In 21 amniotic fluids past the 26th week, 17 cases (85-) had markedly elevated AFP levels; however, 2 cases of anencephaly, 1 of spina bifida, and 1 of hydrocephaly gave levels within the normal range. It is concluded that elevated AFP in the amniotic fluid is a reliable but nonspecific marker for open neural tube defects prior to the 26th week of pregnancy, but may become normal after the 26th week in a small percentage of patients.

PIP: A steady decline in alpha fetoprotein (AFP) levels was observed in single specimens of amniotic fluid (AF) from 237 patients, ranging from 26 mcg/ml at 7-9 weeks to 155 ng/ml at term. All pregnancies tested were normal. 35 AF specimens from 33 confirmed neural tube defective pregnancies were assayed for AFP. Very high levels of AFP were found in 13/14 fluids examined before Week 26 of gestation. A value of 23 mcg/ml was determined in 1 sample where the infant had skin-covered encephalocele. A fluid taken from the same patient at 34 weeks fell to 6.4 mcg of AFP. 21 AF samples from patients past the 26th week of pregnancy were analyzed. Of these, 1 case of spina bifida and 2 of anecephaly gave no detectable levels of AFP by electroimmunodiffusion. By radioimmunoassay, however, these samples measured 3700, 256, and 700 ng/ml. 1 case of hydroencephaly, examined at 33 weeks, had an AFP level of 1.5 mcg/ml. A sharp drop in AFP from 353.6 at 15 weeks to 10.4 mcg/ml at 29 weeks was noted in the only serially examined open neural tube defective pregnancy.