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Biomedical subjects

G H Stein

Publications and source records attributed to G H Stein.

At least 19 recordsLinked to original sources
PubMed1992

A comparison of the safety of therapeutically equivalent doses of isradipine and diltiazem for treatment of essential hypertension.

We compared the safety of a new dihydropyridine calcium entry blocker, isradipine, with an equipotent dose of diltiazem in 174 mild hypertensives (diastolic blood pressure [DBP] 95 to 105 mm Hg). After appropriate washout and placebo periods, patients were randomly assigned to receive either 1.25 mg isradipine twice daily (Group I) or 40 mg diltiazem thrice daily (Group D). If DBP remained above 90 mm Hg, doses were increased to a maximum of 5 mg isradipine twice daily or 120 mg diltiazem thrice daily. Active therapy was given for a total of 12 weeks. Only 18 patients (nine from each group) did not complete the protocol. The patients were well-matched at baseline with a mean BP of 149/100 mm Hg for those who were randomized to isradipine and completed the protocol and 153/99 mm Hg for the diltiazem group. The responses to each drug were excellent with 72% of the isradipine patients and 73% of the diltiazem group having DBP less than 90 mm Hg at the completion of the study. Of the 156 patients who completed the protocol, only 18 patients (ten in Group I and eight in Group D) failed to respond. Both drugs were well-tolerated. No adverse reactions were reported by 68 percent of the patients in Group I and 65% of those in Group D. The most common side effect was headache (9.0% in Group I and 7.8% in Group D) followed by fatigue (5.2% in Group I and 3.9% in Group D). Age and race did not predict response to either agent but men responded slightly better to diltiazem than women. We conclude that isradipine and diltiazem are equally well tolerated and can be used successfully as a monotherapy to treat hypertension in a wide variety of patients.

Adult↗
PubMed1991

Senescent cells fail to express cdc2, cycA, and cycB in response to mitogen stimulation.

Senescent human diploid fibroblasts (HDF) contain no detectable cdc2 mRNA or p34cdc2 protein. Similarly, young quiescent HDF have only low levels of cdc2 mRNA and protein. After serum stimulation, quiescent HDF accumulate increasing amounts of cdc2 mRNA and protein and go through DNA synthesis and mitosis. In contrast, serum-stimulated senescent HDF fail to accumulate detectable amounts of cdc2 mRNA and protein and fail to enter S phase. Mitosis is likewise deficient in senescent cells even when they have been induced to synthesize DNA by simian virus 40 large tumor antigen. Since p34cdc2 or its homologues appear to be required for DNA synthesis and mitosis in eukaryotes, a lack of these molecules in serum-stimulated senescent HDF could be an important reason for their inability to enter S phase or mitosis. Nuclear microinjection of cdc2 DNA into senescent HDF causes rounding up of the cells but no induction of DNA synthesis. Since cyclins A and B are important cofactors of the protein kinase activity of p34cdc2 or its homologues, we analyzed expression of these genes in serum-stimulated senescent HDF and determined that they contain little or no cycA or cycB mRNA. These deficiencies may be relevant to the lack of DNA synthesis and mitosis in senescent HDF.

Amino Acid Sequence↗
PubMed1991

5-Azacytidine-induced demethylation of DNA to senescent level does not block proliferation of human fibroblasts.

IMR-90 human diploid fibroblasts (HDF) lose from 30-50% of their genomic 5-methyldeoxycytidine (5mdC) during the cellular aging process. In contrast, immortal SV40-transformed IMR-90 maintain a constant level of 5mdC in culture. Precrisis SV40-transformed HDF (AG3204) represent a stage in between normal cell aging and immortalization because these cells still have a finite proliferative lifespan, but it is longer than that of normal HDF and ends in cell death rather than in G1-arrest. We find that AG3204 cells continue to lose from 12-33% of their 5mdC after a population has become 99% positive for SV40 T-antigen. Both IMR-90 cells and AG3204 cells have similar levels of 5mdC (average of 2.25%) at the end of lifespan. We investigated whether this level of 5mdC is an absolute block to further proliferation by treating IMR-90 and AG3204 cells with 5-azacytidine (5azaC) to reduce their 5mdC levels below the terminal level normally achieved at end of lifespan. We find that both IMR-90 and AG3204 cells undergo extensive proliferation with subterminal levels of 5mdC and that the lifespans of both cell types are shortened by 5azaC treatment. These studies indicate that random genomic DNA demethylation to a specific level of 5mdC is not a direct cause of finite proliferative lifespan. However, the correlation between accelerated DNA demethylation and accelerated aging still suggests that these two phenomena are related. Two ways to explain this relationship are: (1) DNA demethylation during aging is not random, and/or (2) both DNA demethylation and other independent aging processes cooperate to produce finite lifespan. In both cases, accelerated random DNA demethylation could accelerate aging, but not necessarily in direct relationship to the final genomic level of 5mdC achieved during the normal aging process.

Azacitidine↗
PubMed1991

Nicardipine as antihypertensive monotherapy: positive effects on quality of life.

The effects of nicardipine and propranolol on patients' quality of life were compared during a double-blind, multicentre, parallel, randomised study of hypertension therapy. After a placebo run-in period, the doses for each patient were successfully titrated to reduce supine diastolic blood pressure to less than 90 mmHg with either nicardipine 60 or 90 mg/day (123 patients) or with propranolol 90-240 mg/day (120 patients). Both drugs demonstrated similar efficacy in reducing systolic and diastolic blood pressure. Total duration of therapy ranged from 6-12 weeks. The Nottingham Health Profile questionnaire was used to assess the effect of each treatment on the patients' quality of life. The overall quality of life score for patients on nicardipine showed a tendency toward improvement, while for those on propranolol, the trend was toward overall worsening. The differences between the two treatment groups were statistically significant for males (P = 0.02). The analysis of the separate components of this evaluation demonstrated that physical mobility was reported to be decreased more for the propranolol-treated patients than for the nicardipine-treated patients (P = 0.02). In contrast to the propranolol-treated patients, the nicardipine-treated patients reported improvements for sleep, social life, work, sex life, and for activities related to hobbies and interests. A second questionnaire was used to assess the effects of the therapies on work productivity. Among those patients who worked for pay, more patients treated with propranolol than those treated with nicardipine rated themselves as less productive at work (P = 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1990

Failure to phosphorylate the retinoblastoma gene product in senescent human fibroblasts.

Heterokaryon studies suggest that senescent and quiescent human diploid fibroblasts (HDF) contain a common inhibitor of entry into S phase. DNA synthesis can be induced in senescent and quiescent HDF by fusing them with cells containing DNA viral oncogenes such as SV40 T antigen, adenovirus E1A, or human papillomavirus E7. Both senescent and quiescent HDF contained the unphosphorylated form (p110Rb) of the retinoblastoma protein, a putative inhibitor of proliferation. After serum stimulation, senescent HDF did not phosphorylate p110Rb and did not enter S phase, whereas quiescent HDF phosphorylated p110Rb and entered S phase. These findings, combined with the observations that T antigen, E1A, and E7 form complexes with, and presumably inactivate, unphosphorylated p110Rb, suggest that failure to phosphorylate p110Rb may be an immediate cause of failure to enter S phase in senescent HDF.

Adenovirus Early Proteins↗
PubMed1990

Immortal phenotype of the HeLa variant D98 is recessive in hybrids formed with normal human fibroblasts.

Normal human cells such as human diploid fibroblasts (HDF) have a finite proliferative lifespan in culture. Previous studies have shown that the limited lifespan phenotype is dominant in cell hybrids formed by fusion of HDF to at least 23 different kinds of immortal human cells. However, two independent studies reported that hybrid clones formed by the fusion of HDF to the HeLa variant D98 had unlimited division potential. Those results were potentially very important because they implied that a) there is a dominant mechanism for immortalization of human cells in addition to the well-documented recessive mechanism, and b) a dominant mechanism would lend itself to identification of the immortalizing gene. Consequently, we carried out more detailed studies of the behavior of D98 cells in hybrids. Our results indicate that the majority of D98 x HDF hybrid clones exhibit a clear-cut finite proliferative lifespan phenotype. In addition, these hybrid cell populations often give rise to an immortal focus of cells that can be seen to take over the population of mortal cells at the end of their lifespan. This phenomenon reconciles our data with the previous reports of immortal D98 x HDF hybrid clones and leads us to conclude that D98 cells do not express a dominant immortalizing gene.

Cell Fusion↗
PubMed1990

Multicenter comparison of once- and twice-daily isradipine to hydrochlorothiazide for the treatment of hypertension in elderly patients.

After 8 weeks of isradipine, a twice-a-day dihydropyridine calcium channel blocker, 49% of elderly patients showed a complete response (sitting diastolic blood pressure less than or equal to 85 mm Hg) and 36% showed a partial response (sitting diastolic blood pressure decrease greater than or equal to 10 mm Hg) for an 85% total response rate. Hydrochlorothiazide gave a complete response in 36% of the patients and a partial response in 33%, for a 69% total response rate (p less than 0.0046). Because elderly subjects have reduced clearance for many drugs, we determined how those who responded to twice-a-day administration would respond to once-a-day administration. After 4 weeks of isradipine administered once a day, 54% of the patients showed a complete or partial response, whereas 38% of the patients who were changed to placebo showed a response. In contrast, 82% of patients receiving hydrochlorothiazide once a day showed a response, whereas 60% of patients who were changed to placebo showed a response. These data indicate that the standard formulation of isradipine was not effective when administered once a day.

Aged↗
PubMed1990

Long-term lipid profiles with isradipine and hydrochlorothiazide treatment in elderly hypertensive patients.

Lipid profiles were determined in 56 elderly patients with benign essential hypertension during an open-label 1-year study of the safety and efficacy of isradipine, a new calcium antagonist, in controlling blood pressure. Patients with diastolic blood pressures between 96 and 115 mm Hg were titrated with isradipine (mean dose of 11 mg/day) to reduce blood pressure to less than 90 mm Hg. Ten of these patients received concomitant hydrochlorothiazide (HCTZ) 50 mg/day for additional control. Sera were analyzed using standard methods at the end of a 2- to 4-week washout period, and at the end of Months 6 and 12, for total cholesterol (CHOL) and HDL- and LDL-cholesterol. Changes in lipid values (mg/dl) from baseline to 12 months with isradipine alone (n = 38) were as follows: CHOL, -7.5; HDL, +3.9 (p less than 0.05); LDL, -6.2; CHOL/HDL, -0.6 (p less than 0.05). For patients receiving concomitant HCTZ (n = 9), the changes were as follows: CHOL, -4.9; HDL, +3.4; LDL, -16.8; CHOL/HDL, -0.4. In conclusion, isradipine alone was associated with significant improvements in HDL cholesterol and total CHOL/HDL ratio. Lipid profiles of patients receiving isradipine and HCTZ were minimally affected. Favorable lipid changes with isradipine suggest that it may have advantages in the treatment of hypertensive patients.

Aged↗
PubMed1989

Coordinate control of growth arrest states in normal human diploid fibroblasts: effect of cloned SV40 tumor antigen genes.

The growth arrest states of quiescence and senescence in normal human diploid fibroblasts (HDF) are related but distinct phenomena. In an effort to (1) understand the extent to which arrest in the quiescent state and arrest in the senescent state share common regulatory mechanisms, and (2) test our hypothesis that when the quiescent phenotype is altered in HDF, then the senescent phenotype is likewise altered, we have transfected HDF with cloned SV40 tumor antigen genes. Introduction of the tumor antigen genes results in a loss of the ability to enter both the quiescent arrest state and the senescent arrest state but does not immortalize the cells or create significant aneuploidy.

Antigens, Polyomavirus Transforming↗
PubMed1988

Human microvascular endothelial cells: coordinate induction of morphologic differentiation and twofold extension of life span.

Human microvascular endothelial cells (HMVEC) from adult adipose tissue were cultured in MCDB 131 medium supplemented with 10% fetal bovine serum. Under these conditions, HMVEC from seven different donors had finite proliferative life spans ranging from 14.5 to 23.5 population doublings (PD), with a mean life span of 19 PD. Addition of 10% conditioned medium from activated human leukocyte cultures (BM Condimed) extended the life span of HMVEC to 31 to 41 PD, with a mean life span of 37 PD. At the end of lifespan, HMVEC cultures both with and without BM Condimed had very low labeling indices (0 to 5% [3H]thymidine labeled nuclei) and consisted of enlarged cells. However, the morphologies of the two types of HMVEC cultures were very different. Untreated HMVEC were polygonal endothelial cells that formed cobblestonelike monolayers with no cell overlapping. In contrast, BM Condimed-treated HMVEC were more elongated, less regularly shaped cells that were not strictly inhibited from overlapping. When old, these cells accumulated numerous vacuoles. The BM Condimed-treated HMVEC expressed Factor VIII antigen, which confirms their identity as endothelial cells. These cells reverted rapidly to the polygonal morphology of untreated HMVEC when they were removed from BM Condimed. Likewise, their proliferative capacity was not extended further once BM Condimed was removed. These results suggest that HMVEC can exist in two distinct morphologic states in which the cells have different finite proliferative life spans.

Cell Differentiation↗
PubMed1988

Comparative evaluation of enalapril and hydrochlorothiazide in elderly patients with mild to moderate hypertension.

Initial treatment of elderly hypertensive patients with an angiotensin-converting enzyme inhibitor is currently discouraged due to such patients' typical low-renin profile. To validate this principle, we studied 38 elderly males (aged greater than or equal to 65 years) with mild to moderate hypertension, comparing hemodynamic responses to and subjective impressions of enalapril or hydrochlorothiazide (HCTZ). After gradual withdrawal of existing antihypertensive therapy and a four-week, single-blind placebo period, each patient was randomized in a double-blind fashion to receive either enalapril 10-20 mg/d or HCTZ 12.5-25 mg/d for two to four weeks. Combination therapy with both agents was employed if either alone failed to reduce seated diastolic BP to less than or equal to 90 mm Hg. Equivalent proportions of patients receiving enalapril or HCTZ (8 of 19 and 10 of 19, respectively; p = ns) responded with significant reductions in systolic and diastolic BP in seated and standing positions. Combination therapy was most effective in patients receiving HCTZ prior to enalapril. In patients receiving enalapril before HCTZ, BP changes were minimal. No adverse effects were observed in the enalapril group but occurred in an equivalent fraction of patients in the other groups (4 of 10 HCTZ alone, 6 of 20 enalapril + HCTZ; p = ns). We conclude that enalapril may be considered a reasonable monotherapeutic antihypertensive agent in some elderly patients. Combination with HCTZ is beneficial in patients who fail to respond adequately to HCTZ alone.

Aged↗
PubMed1988

High efficiency DNA-mediated transformation of human diploid fibroblasts.

Normal human diploid fibroblasts (HDF) have only rarely been used as recipients in DNA transfection experiments even though they have the potential to add to our knowledge about the genes that control normal cell proliferation. A major impediment to the use of HDF has been their poor transfection frequency. In this paper, we show that IMR-90 human diploid fibroblasts can be stably transfected at high efficiency with pRSVneo at an average frequency of 2 x 10(-3) (0.2%) using a modified calcium phosphate-DNA coprecipitation transfection protocol. Prior to this report, the best transfection frequencies of HDF were on the order of 10(-5)-10(-4). In the present protocol it is very important to maintain relatively low cell densities, particularly during the selection process post-transfection. Using this protocol other neo-containing plasmids also yield high transfection frequencies in IMR-90. IMR-90, which are readily available from the NIH Aging Cell Repository, may be one of the best HDF to use as recipients because they are five times as efficient as are WI-38, which is another well characterized HDF.

Calcium Phosphates↗
PubMed1986

Quiescent human diploid fibroblasts. Common mechanism for inhibition of DNA replication in density-inhibited and serum-deprived cells.

The mechanism for cessation of proliferation in density-inhibited quiescent human diploid fibroblasts (HDF) and serum-deprived quiescent HDF was compared in two ways. Density-inhibited HDF were fused to either replicating HDF or SV40-transformed HDF and DNA synthesis was measured in the resulting heterokaryons. DNA synthesis was inhibited in the replicating HDF nuclei in heterokaryons in a way that suggested that entry into S phase was blocked, but ongoing DNA synthesis was not inhibited. In contrast, DNA synthesis was induced in the quiescent nuclei in heterokaryons formed with SV40-transformed HDF. Previous experiments had shown that serum-deprived HDF also behave in this way in heterokaryons. To test this similarity further, we examined the inhibitory activity of cell membranes prepared from both types of quiescent HDF. We found that both types of quiescent HDF contain DNA synthesis-inhibitory activity that is (1) effective on replicating HDF; (2) ineffective on SV40-transformed HDF; (3) sensitive to heat and trypsin. Thus, these results support the hypothesis that both density-inhibited HDF and serum-deprived HDF share a common mechanism for arrest in G1 phase. They also suggest that a membrane-bound protein plays a role in the inhibition of DNA synthesis in quiescent HDF.

Blood↗
PubMed1986

Membrane-associated inhibitor of DNA synthesis in senescent human diploid fibroblasts: characterization and comparison to quiescent cell inhibitor.

Cell membranes prepared from senescent human diploid fibroblasts (HDF) inhibited entry into S phase by 35% when added to the medium of replicating young HDF. This membrane-associated inhibitory activity was (i) sensitive to trypsin, heat, and periodate, which suggests that the inhibitor is a glycoprotein, and (ii) not able to inhibit DNA synthesis in simian virus 40-transformed HDF, which indicates that not all types of cells are sensitive to this inhibitor. Quiescent young HDF also have a surface membrane-associated inhibitor of DNA synthesis. A comparison of the senescent HDF and quiescent HDF inhibitor activities indicates that they may have the same chemical and physical nature and the same specific activity, but their regulation is different. The inhibitory activity of quiescent young HDF is abolished within 20 hr after refeeding with fresh serum-containing medium, whereas that of senescent HDF remains unchanged. Quiescent old HDF (two or three population doublings remaining) exhibit an intermediate response to serum with approximately two-thirds of the inhibitory activity abolished. The fraction of cells in S phase at 20-24 hr post-stimulation (37% in young HDF, 24% in old HDF, and 0% in senescent HDF) is inversely proportional to inhibitor levels. This suggests that inability to neutralize the inhibitory activity in response to serum stimulation could be involved in the inability of senescent HDF to enter S phase. Disappearance of the inhibitory activity from quiescent young HDF occurs late in G1 phase. Thus, the inhibitor may play a role in determining the length of the G0 to S phase transition in these cells.

Cell Cycle↗
PubMed1985

Relationship of finite proliferative lifespan, senescence, and quiescence in human cells.

Cell hybrids were formed between human diploid fibroblasts (HDF) and carcinogen-transformed HDF to determine the relationship among: (1) finite proliferative lifespan, which we define as an age-related failure of a population to achieve one population doubling in 4 weeks; (2) arrest in a senescent state, which we define as cessation of DNA synthesis in a viable culture that is at the end of its lifespan by the above definition; and (3) arrest in a quiescent state, which we define as cessation of DNA synthesis in a young culture that is crowded or mitogen-deprived. HDF express all three of these phenotypes, which we have abbreviated FPL+, S+, and Q+, respectively. Carcinogen-transformed HDF are transformed to immortality (FPL-) and inability to achieve quiescence (Q-). They have no S phenotype because, by definition, this phenotype only exists in FPL+ cells. Fusion of FPL+, Q+, S+ HDF X FPL-, Q- carcinogen-transformed HDF produced hybrid clones that were FPL+, Q-, and S-, where the S- phenotype means that individual cells continued to synthesize DNA in cultures that had reached the end of their lifespan by our definition. These results are consistent with our hypothesis that senescent HDF and quiescent HDF may share a common mechanism for arrest in G1 phase. We have suggested that this could occur if the aging mechanism that is responsible for the FPL+ phenotype is a progressive decrease in the ability of cells to recognize or respond to mitogenic growth factors. If so, then cells would become physiologically mitogen-deprived at the end of their lifespan, which would cause them to arrest in the senescent state by the same mechanism that causes young cells to arrest in the quiescent state when they are mitogen-deprived. This hypothesis predicts that the FPL+ phenotype can be separated from the S+ phenotype--i.e., FPL+ cells can be S+ or S- --and that the Q and S phenotypes are linked--i.e., FPL+ cells are either Q+ and S+ or Q- and S-. Both these predictions are supported by the present data.

Cell Division↗
PubMed1985

SV40-transformed human fibroblasts: evidence for cellular aging in pre-crisis cells.

Pre-crisis SV40-transformed human diploid fibroblast (HDF) cultures have a finite proliferative lifespan, but they do not enter a viable senescent state at end of lifespan. Little is known about either the mechanism for this finite lifespan in SV40-transformed HDF or its relationship to finite lifespan in normal HDF. Recently we proposed that in normal HDF the phenomena of finite lifespan and arrest in a viable senescent state depend on two separate processes: 1) an age-related decrease in the ability of the cells to recognize or respond to serum and/or other mitogens such that the cells become functionally mitogen-deprived at the end of lifespan; and 2) the ability of the cells to enter a viable, G1-arrested state whenever they experience mitogen deprivation. In this paper, data are presented that suggest that pre-crisis SV40-transformed HDF retain the first process described above, but lack the second process. It is shown that SV40-transformed HDF have a progressively decreasing ability to respond to serum as they age, but they continue to traverse the cell cycle at the end of lifespan. Concomitantly, the rate of cell death increases steadily toward the end of lifespan, thereby causing the total population to cease growing and ultimately to decline. Previous studies have shown that when SV40-transformed HDF are environmentally serum deprived, they likewise exhibit continued cell cycle traverse coupled with increased cell death. Thus, these results support the hypothesis that pre-crisis SV40-transformed HDF still undergo the same aging process as do normal HDF, but they end their lifespan in crisis rather than in the normal G1-arrested senescent state because they have lost their ability to enter a viable, G1-arrested state in response to mitogen deprivation.

Blood↗
PubMed1979

T98G: an anchorage-independent human tumor cell line that exhibits stationary phase G1 arrest in vitro.

T98 and T98G are two related cell lines that were derived from a human glioblastoma multiforma tumor. T98G has almost twice as many chromosomes as T98, suggesting that it is a polyploid variant of T98. Three aspects of control of cellular proliferation were studied in T98 and T98G cells in comparison to WI-38 normal human diploid cells. WI-38 cells have the following properties: (1) they can undergo only a limited number of population doublings in vitro; (2) they cannot proliferate without anchorage; and (3) they become arrested in G1 phase under stationary phase conditions. T98 cells differ from normal cells in all three of these properties, as do many other transformed cell lines. However, the derivative of T98, namely T98G, expresses an unique combination of normal and transformed aspects of the control of cellular proliferation. T98G cells are like normal cells in that they become arrested in G1 phase under stationary phase conditions, yet they also exhibit the transformed characteristics of anchorage independence and immortality. Thus, T98G cells demonstrate that transformation to immortality and anchorage independence can exist without concomitant loss of the normal mechanism for G1 arrest in response to stationary phase conditions. This result supports the hypothesis that each of these three aspects of control of cellular proliferation can be altered independently. Partially transformed cell lines, such as T98G, should be useful for sorting out the biochemical changes associated with transformation in each of these aspects.

Cell Cycle↗